Endogenous repair factor production promoters

ABSTRACT

It relates to an endogenous repair factor production accelerator which comprises one or at least two selected from prostaglandin (PG) I2 agonist, EP2 agonist and EP4 agonist. Since prostaglandin (PG) I2 agonist, EP2 agonist or EP4 agonist has various endogenous repair factor production accelerating action, angiogenesis acceleration action and stem cell differentiation induction action, it is useful as preventive and/or therapeutic agents for ischemic organ diseases (e.g., arteriosclerosis obliterans, Buerger disease, Raynaud disease, myocardial infarction, angina pectoris, diabetic neuropathy, spinal canal stenosis, cerebrovascular accidents, cerebral infarction, pulmonary hypertension, bone fracture, Alzheimer disease, etc.) and various cell and organ diseases.

TECHNICAL FIELD

The present invention relates to an endogenous repair factor productionaccelerator which comprises one or at least two selected fromprostaglandin (hereinafter referred to as “PG”) I2 agonist, EP2 agonistand EP4 agonist.

BACKGROUND ART

Regeneration medical treatment is drawing attention as the regenerationtherapy at the time of disorders of tissues and cells such as bloodvessel, liver, kidney, lung, pancreas, bone, skeletal muscle cell,myocardial cell, peripheral and central nerve cells and the like. Theself repair system includes a system which is attained by the celldivision of mature cells (simple duplication system) like theregeneration of many parenchymal organs such as liver and kidney and asystem mediated by the proliferation and differentiation induction ofstem cells (precursor cells) (stem cell system) like the regeneration ofhematopoietic cells. Presently, it is said that these two systems arepresent in the regeneration of many tissues and organs. For example, itis said that these two systems are present also in the angiogenesis(regeneration), and there are an angiogenesis system based on the growthof neighboring vascular endothelial cells, vascular smooth muscle cellsand the like, effected by the release of various endogenous repairfactors (e.g., vascular endothelial growth factor (VEGF), hepatocytegrowth factor (HGF), various fibroblast growth factors (a/b FGF),transformation growth factor-β (TGF-β), platelet derived growth factor(PDGF), angiopoietin, hypoxia inducing factor (HIF), insulin-like growthfactor (IGF), bone morphogenetic protein (BMP), connective tissue growthfactor (CTGF), epidermal growth factor (EGF), etc., growth factors oftheir families, and the like) from (vascular) endothelial cells,(vascular) smooth muscle cells, fibroblasts, synovial cells, epithelialcells, platelets, monocytes, lymphocytes, macrophages and the like ofthe injured (neighboring) region, and a vasculogenesis system in whichblood vessel is formed by the differentiation induction of vascularendothelial stem cells from bone marrow cells of a matured individual,effected by the release of various inflammatory cytokines (e.g., IL-1,IL-4, IL-8, TNFα, IFNα/γ, G-CSF, GM-CSF, NO (nitric monoxide), etc.),and various endogenous repair factors.

Regarding the presence of stem cells (precursor cells), they are presentnot only in blood vessels but also in many tissues such as hepatocyte,pancreatic (,) cell, myocardial cell, kidney, lung, bone, joint, nerve,fat, skin and the like, and are proliferated and differentiation-inducedby various endogenous repair factors, various inflammatory cytokines andthe like.

When production of these endogenous repair factors is accelerated,formation of collateral circulation passage is accelerated by the effectof angiogenesis on the ischemic region. Also, it is known thatprevention and treatment (repairing regeneration) of various organdisorders are accelerated by the differentiation induction action fromvarious tissue stem cells. For example, it is known that HGF has a cellgrowth acceleration action, a morphogenesis inducing action, adifferentiation inducing action, a wandering acceleration action,anti-apoptosis action and the like (e.g., see Biochern. Biophys. Res.Commun., 239, 639-644 (1997), etc.). It is known that these endogenousrepair factor production accelerations are effective for the preventionand/or treatment of, for example, liver diseases (e.g., fluminanthepatitis, acute hepatitis, hepatic cirrhosis, fatty liver, livertransplantation, etc.), kidney diseases (e.g., acute renalinsufficiency, chronic renal insufficiency, etc.), lung diseases (e.g.,acute pneumonia, pulmonary fibrosis, pulmonary hypertension, chronicobstructive pulmonary disease (COPD), asthma, etc.), pancreas diseases(e.g., diabetes mellitus, chronic pancreatitis, etc.), bone diseases(e.g., osteoarthritis, articular rheumatism, osteoporosis, bonefracture, periosteum injury, etc.), digestive organ diseases (e.g.,gastric ulcer, duodenal ulcer, ulcerative colitis, Crohn disease, etc.),nerve degeneration diseases (e.g., stroke, Parkinson disease, Alzheimerdisease, spinal canal stenosis, cerebrovascular accidents, moyamoyadisease, etc.), diabetic complications (e.g., nerve disorder, skinulcer, nephropathy, retinal disease, etc.), vascular endothelial celldiseases (e.g., restenosis after PTCA (percutaneous transluminalcoronary angioplasty), arteriosclerosis, etc.), heart diseases (e.g.,supraventricular tachyarrhythmia, congestive heart failure, coronaryartery disease, sudden cardiomyopathy, dilated cardiomyopathy, etc.),dental diseases (e.g., periodontal disease, tooth extraction wound, oralwound, periodontal tissue disease, etc.), decubitus, glaucoma, alopeciaand the like.

The regeneration therapy mediated by endogenous repair factors isdrawing attention as an angiogenesis (regeneration) therapy and a tissuegeneration therapy at the time of the diseases of organs and tissuessuch as liver, pancreas, kidney, heart, central/peripheral nerves, bloodvessel, tooth, eye, periosteum, bone and the like. This is drawingattention particularly as a regeneration therapy of serious ischemicorgan diseases having no therapeutic methods, and several methods areexamined on arteriosclerosis obliterans (hereinafter referred to as“ASO”), Buerger disease, Raynaud disease, cardiovascular diseases (e.g.,myocardial infarction, angina pectoris, etc.), diabetic neuropathy,spinal canal stenosis, ischemic brain disease (e.g., cerebrovascularaccidents, cerebral infarction, etc.), pulmonary hypertension, bonefracture, or Alzheimer disease and the like.

For example, patients of obstructive peripheral blood vessel diseasestypified by ASO and Buerger disease show intermittent claudication, painduring resting and ulcer and necrosis of the legs, and finally itbecomes unavoidable to undergo amputation of the legs. However, atpresent, there is no therapeutic method effective for these serious ASOpatients. Since intravenous injection of PGE 1 and a vasodilator orplatelet agglutination inhibitor as an oral agent of cilostazol do notshow their effects on serious ASO patients, an intravascular treatment(balloon dilation or Stent insertion) and revascularization cannot becarried out. Recently, a gene therapy in which a VEGF gene plasmid andan HGF gene plasmid are directly administered by intramuscular injectioninto skeletal muscles of ischemic regions of the legs of these patients(cf, Circulation, 97, 1114-1123 (1998) and Gene Therapy;, 8, 181-189(2001)) has been clinically applied, and its effect is drawingattention. In addition, slow release preparations of a growth factorprotein (e.g., a gelatin inclusion sheet) have also been subjected tobasal examinations (Circulation, 106, Supple 2, II 350 (2002)).

On the other hand, an angiogenesis therapy in which vascular endothelialstem cells separated from bone marrow or peripheral blood of a patientare directly administered into the ischemic region of a leg byintramuscular injection is drawing attention and is carried out atseveral university hospitals which is also drawing attention (cf. THELANCET, 360, 427-435 (1002)).

Introduction of these genes and stem cells directly into ischemicregions using a vascular catheter equipped with a low invasion needlebecame possible also in myocardial infarction and angina pectoris, andis under clinical application as an angiogenesis therapy. For example,it has been reported that ischemia is improved for unstable angina byinjecting a VEGF gene plasmid directly into heart muscle (Circulation,98, 2800-2804 (1998)). Also, it was considered that PDGF is concerned inthe angiogenesis after stroke, because its increase was observed innerve cell of the phenanbla (the tissue is not dead by infarction butcannot perform its function) region of infarct focus of a cerebralinfarction patient (Stroke, 28, 564-573 (1997)). Gene therapy and thelike using a vector to the brain via cerebrospinal fluid fromcerebellomedullary cistern have also been attempted on the ischemiccerebrovascular accidents. These treatments are a therapeuticangiogenesis (regeneration) therapy which prompts development ofcollateral circulation passage to the ischemic region and is a tissueregeneration therapy by the differentiation induction of tissue stemcells. However, clinical application of these gene therapy and celltherapy have many problems in terms of ethics, safety (immunity,infection, cancer, etc.), flexibility, economy and the like.

As a reopening therapy at the time of blood vessel obstruction in ASO,myocardial infarction, angina pectoris, arteriosclerosis and the like,PTCA (percutaneous transluminal coronary angiopathy) has been carriedout with good results. However, it is known that restenosis is inducedby the injury of vascular endothelial cells around the obstruction dueto forced vasodilation by balloon dilation, Stent indwelling and thelike. As a method for preventing restenosis, a platelet agglutinationinhibitor or the like is administered, but this is still anunsatisfactory state. Recently, drugs such as antibiotics andcarcinostatic agents (rapamycin, sirolimus, etc.) and a Stent coatedwith a radioisotope preparation such as β rays have been developed withgood results on the prevention of restenosis (N. Eng. J. Med., 346(23),1769-1771 (2002)). However, these methods also have many problems from along-term point of view. Accordingly, concern is directed toward amedicament which enhances platelet agglutination inhibitory action attopical injured regions of vascular endothelial cells and damagerepairing action by endothelial cell growth.

On the other hand, prostaglandin (PG) is a natural physiologicallyactive substance biosynthesized from PGH2 in a metabolic pathway in theliving body, which is called arachidonic acid cascade. The biosynthesisenzymes from arachidonic acid to PGH2 are called cyclooxigenase (COX),and COX-1, COX-2 and COX-3 are known at present (Proc. Natl. Acad. Sci.,99, 1371 (2002)). In addition, compounds which inhibit these enzymes aregenerally used as antipyretic, analgesic and anti-inflammatory agentsand agents for preventing circulatory organ system diseases, asnon-steroidal anti-inflammatory drug (NSAID). Particularly, COX-2induced in inflammation regions is concerned in the biosynthesis ofPGI2, PGE2 and the like. These biosynthesized PGs are concerned in theonset of pyrexia, pain and inflammation in the inflammation regions andhealing thereof That is, the PGs biosynthesized at the inflammationregions directly act as inflammation-causing agents, induce variousinflammatory cytokines, evoke inflammation, and accelerate healingthereof

On the other hand, it is known that patients who took NSAID for aprolonged period of time have significantly low mortality rate by largebowel cancer and lung cancer (N. Eng. J. Med., 328, 1313-1316 (1993)).It is said that this action has a cancer cell growth inhibitory action,because angiogenesis for cancer tissue growth is inhibited through thebiosynthesis inhibition of PGI2, PGE2 and the like by NSAID. That is, ananti-angiogenesis therapy which controls growth and metastasis of tumorsby inhibiting angiogenesis is drawing attention as a new strategy ofcancer treatment. Also, it is known that a COX-2-selective inhibitordoes not induce gastric ulcer when it is administered, but prolongshealing of gastric ulcer. There is a report stating that its cause isinhibition of angiogenesis action for repairing of injured tissues (Am.J. Med., 104, 43S-51S (1998)). In addition, selective COX-2 inhibitorscontrol the angiogenesis accompanied by inflammation (Jpn. J.Pharmacol., 75, 105-114 (1997)).

It is known that PGI2 has a markedly strong platelet agglutinationinhibitory action, as well as actions such as platelet adhesion,vasodilation and gastric acid secretion inhibition. In addition, PGE2administration accelerates accumulation of inflammatory cells includingmonocyte and production of inflammatory cytokine (e.g., IL-1(interleukin-1), IL-8, IL-6, IFN-α (interferon-α), TNFα (tumor necrosisfactor α), and NO (nitric monoxide), etc.), and acts as aninflammation-causing agent.

JP-A-6-87811 discloses in its specification that the oxime derivativerepresented by formula (I) which is described later or a non-toxic saltthereof, to be used in the present invention as a PGI2 agonist (IPagonist), is useful for the prevention and/or treatment of thrombosis,arteriosclerosis, ischemic heart disease, gastric ulcer, hypertensionand the like, because it has platelet agglutination inhibition, plateletadhesion inhibition, vasodilation and gastric acid secretion inhibitionactions, but it does not describe or suggest on the angiogenesis actionby exerting vascular endothelial cell and vascular smooth muscle cellgrowth action based on the endogenous repair factor productionacceleration action, and on the various cell and organ diseases (theabove-described diseases to be prevented and treated (repairregeneration) by HGF) by differentiation induction of various stem cellscaused by these endogenous repair factor production acceleration actionand the like.

Also, it is reported in Diabetologia., 40, 1053-1061 (1997) that a PGI2derivative Beraprost ((±)-(1R,2R,3aS,8bS)-2,3,3a,8b-terahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butanoicacid sodium salt) increases HGF production in vascular endothelial cellsin vitro and shows endothelial cell growth action, but there are nodescriptions on the angiogenesis accelerating action by the topicaladministration of persistent preparation of Beraprost and on itsusefulness for the prevention and treatment of various organ diseases.

In addition, PGE2 is known as a metabolic product in the arachidonicacid cascade, and it is known that its actions have various functionssuch as cell protection action, uterine contraction action, painproducing action, digestive organ peristalsis acceleration action,stimulation action, gastric acid secretion inhibition action, bloodpressure reducing action, diuretic action and the like.

From the studies in recent years, it has been revealed that subtypeshaving respectively different roles are present in the PGE2 receptor.The subtypes known at present are roughly divided into four, and arerespectively called EP1, EP2, EP3 and EP4 (J. Lipid Mediators CellSignaling, 12, 379-391 (1995)). By examining their share of roles andthereby finding a compound which does not bind to other subtypereceptors, it became possible to obtain a medicament having less sideeffects.

For example, JP-A-11-193268 discloses in its specification that acompound represented by formula (I-a), a non-toxic salt thereof, or aprodrug or cyclodextrin clathrate thereof, which is used in the presentinvention as an EP2 agonist, is useful in preventing and/or treatingimmune diseases (e.g., autoimmune disease, organ transplantation, etc.),asthma, osteogenesis abnormality, nerve cell death, hepatopathy,premature delivery, abortion, retinal nerve diseases such as glaucoma,and the like, but it does not describe or suggest on the endogenousrepair factor releasing action, stem cell differentiation inductionaction and angiogenesis acceleration action.

For example, WO00/03980 discloses in its specification that a compoundrepresented by formula (I-b), a non-toxic salt thereof, or acyclodextrin clathrate thereof, which is used in the present inventionas an EP4 agonist, is useful in preventing and/or treating diseasesincluding immune diseases (amyotrophic lateral sclerosis (ALS), multiplesclerosis, Sjoegren syndrome, rheumatoid arthritis, autoimmune diseasessuch as systemic lupus erythematosus, rejection after organtransplantation, etc.), asthma, osteogenesis abnormality, nerve celldeath, lung disease, hepatopathy, acute hepatitis, glomerulonephritis,renal insufficiency, hypertension, myocardial ischemia, systemicinflammatory reaction syndrome, burn pain, sepsis, hemophagocytosissyndrome, macrophage activation syndrome, Still disease, Kawasakidisease, burn injury, systemic granuloma, neoplastic colitis, Crohndisease, hypercytokinemia at the time of dialysis, multiple organfailure, shock, sleep abnormality, platelet agglutination and the like,but it does not describe or suggest on the endogenous repair factorreleasing action, stem cell differentiation induction action andangiogenesis acceleration action.

DISCLOSURE OF THE INVENTION

Great concern has been directed toward the development of an endogenousrepair factor production accelerator, a stem cell differentiationinducer (precursor cell differentiation inducer) and an angiogenesisaccelerator, which are useful as preventive and therapeutic agents of(ischemic) organ diseases and have less side effects.

With the aim of finding an endogenous repair factor productionaccelerator, a stem cell differentiation inducer and an angiogenesisaccelerator, which are useful as preventive and therapeutic agents of(ischemic) organ diseases, the present inventors have conductedintensive studies and found as a result that a PGI2 agonist (e.g., acompound represented by formula (I) or a salt thereof), an EP2 agonist(e.g., a salt represented by formula (I-a), a prodrug thereof or acyclodextrin clathrate thereof) or an EP4 agonist (e.g., a compoundrepresented by formula (I-b), a salt thereof or a cyclodextrin clathratethereof) can achieve the object, thereby accomplishing the presentinvention.

Also, the inventors have considered that when a PGI2 agonist, an EP2agonist or an EP4 agonist could be topically administered to an ischemicregion requiring angiogenesis or a injured region requiring tissuerepair, it will become possible to produce an endogeneous repair factor,an inflammatory cytokine and the like in the periphery of the injuredtopical region, in addition to the vasodilation of the remaining bloodvessels in the ischemic region and platelet agglutination inhibitoryaction, so that a medicament having less side effects in systemicadministration could be created. In addition, it was considered thatwhen it is possible to produce a pharmaceutical preparation forpersistent release during a period until angiogenesis (regeneration) ortissue repair is effected in the ischemic region or periphery of thetissue injury topical region, a medicament having less side effects insystemic administration and with improved administration compliance ofsmall administration frequency could be created.

For example, angiogenesis requires a period of generally from 1 week to6 months, more preferably from 1 week to 8 weeks, and persistent releaseof the active ingredient is required in the ischemic region during theperiod. In addition, since PGI2 agonists, EP2 agonists and EP4 agonistsshow vasodilation action and platelet agglutination inhibitory action inaddition to angiogenesis acceleration action and vascular stem celldifferentiation induction action when production of various endogenousgrowth factors is accelerated, it was considered that they will showfurther strong preventive and/or therapeutic effect upon ischemic organdiseases when these actions are added.

Thus, the inventors have also found as a result of intensive studiesthat a persistent preparation of a PGI2 agonist, an EP2 agonist or anEP4 agonist can achieve the above-described object, thus accomplishingthe present invention.

That is, the present invention relates to the followings:

(1) An endogenous repair factor production accelerator, which comprisesone or at least two selected from a PGI2 agonist, an EP2 agonist and anEP4 agonist.

(2) The endogenous repair factor production accelerator according to theabove (1), wherein the endogenous repair factor is a vascularendothelial growth factor, a hepatocyte growth factor, a fibroblastgrowth factor, a transformation growth factor-β, a platelet derivedgrowth factor, a bone morphogenetic protein or an epidermal growthfactor.

(3) The endogenous repair factor production accelerator according to theabove (1), which is a stem cell differentiation inducer.

(4) The endogenous repair factor production accelerator according to theabove (1), which is an angiogenesis accelerator.

(5) The endogenous repair factor production accelerator according to theabove (1), which is a persistent preparation which further comprises abiodegradable polymer.

(6) The endogenous repair factor production accelerator according to theabove (5), wherein the persistent preparation is a microspherepreparation, a microcapsule preparation or a nanosphere preparation.

(7) The endogenous repair factor production accelerator according to theabove (1), which is an agent for preventing and/or treating organdiseases.

(8) The endogenous repair factor production accelerator according to theabove (7), wherein the organ disease is an ischemic organ disease, aliver disease, a kidney disease, a lung disease, a pancreas disease, abone disease, a digestive organ disease, a nerve degeneration disease, adiabetic complication, a vascular endothelial cell disease, a heartdisease, a dental disease, decubitus, glaucoma or alopecia.

(9) The endogenous repair factor production accelerator according to theabove (8), wherein the ischemic organ disease is arteriosclerosisobliterans, Buerger disease, Raynaud disease, myocardial infarction,angina pectoris, diabetic neuropathy, spinal canal stenosis,cerebrovascular accidents, cerebral infarction, pulmonary hypertension,bone fracture or Alzheimer disease.(10) The endogenous repair factor production accelerator according tothe above (1), wherein the PGI2 agonist is a compound represented byformula (I):

wherein R¹ represents hydrogen or C1-4 alkyl;

R² represents (i) hydrogen, (ii) C1-8 alkyl, (iii) phenyl or C4-7cycloalkyl, (iv) a 4- to 7-membered monocyclic ring containing onenitrogen atom, (v) C1-4 alkyl substituted with a benzene ring or C4-7cycloalkyl, or (vi) C1-4 alkyl substituted with a 4- to 7-memberedmonocyclic ring containing one nitrogen atom;

R³ represents (i) Cl-8 alkyl, (ii) phenyl or C4-7 cycloalkyl, (iii) a 4-to 7-membered monocyclic ring containing one nitrogen atom, (iv) C1-4alkyl substituted with a benzene ring or C4-7 cycloalkyl, or (v) C1-4alkyl substituted with a 4- to 7-membered monocyclic ring containing onenitrogen atom;

e represents an integer of from 3 to 5;

f represents an integer of from 1 to 3;

p represents an integer of from 1 to 4;

r represents an integer of from 1 to 3;

q represents an integer of 1 or 2, and

wherein, when

is the group represented by (iii) or (iv),

-(CH₂)_(p)- and ═CH—(CH₂)_(s)- are bound to the position of a or b onthe ring, and

the rings in R² and R³ may be substituted with 1 to 3 of C1-4 alkyl,C1-4 alkoxy, halogen, nitro or trihalomethyl, or

a salt thereof

(11) The endogenous repair factor production accelerator according tothe above (10), wherein the PGI2 agonist is

(1)(E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid, or

(2)(Z)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid.

(12) The endogenous repair factor production accelerator according tothe above (1), wherein the PGI2 agonist is

(1) (±)-(1R,2R,3 aS,8bS)-2,3,3a,8b-terahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofiiran-5-butanoicacid sodium salt,

(2) 5-{(3aR,4R,6aS)-5-hydroxy-4-[(1E,3S)-3-hydroxy-3-(cis-4-propylcyclohexyl)prop-1-enyl-3,3a,4,5,6,6a-hexahydrocyclopenta[b]pyrrol-2-yl}pentanoicacid methyl ester, or

(3)(5E)-5-[(3aS,4R,5R,6aS)-4-[(1E,3S)-3-cyclopentyl-3-hydroxyprop-1-enyl]-5-hydroxyhexahydropentalene-2(1H)-ylidene]pentanoicacid.(13) The endogenous repair factor production accelerator according tothe above (1), wherein the EP2 agonist is a compound represented byformula (I-a):

wherein R^(a) represents carboxyl or hydroxymethyl;

R^(1a) represents oxo, methylene or halogen;

R^(2a) represents hydrogen, hydroxyl or C1-4 alkoxy;

R^(3a) represents hydrogen, C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, orC1-8 alkyl, C2-8 alkenyl or C2-8 alkynyl substituted with 1 to 3 of thefollowing groups (1) to (5): (1) halogen, (2) C1-4 alkoxy, (3) C3-7cycloalkyl, (4) phenyl, (5) phenyl substituted with 1 to 3 halogen, C1-4alkyl, C1-4 alkoxy, nitro or trifluoromethyl;

na represents 0 or an integer of from 1 to 4;

represents a single bond or a double bond;

represents a double bond or a triple bond; and

represents a single bond, a double bond or a triple bond, and

wherein (1) when the 5-6 position represents a triple bond, the 13-14position does not represent a triple bond, and

(2) when the 13-14 position represents a double bond, the a double bondrepresents E form, Z form or EZ form,

a salt thereof, a prodrug thereof or a cyclodextrin clathrate thereof

(14) The endogenous repair factor production accelerator according tothe above (13), wherein the EP2 agonist is (5Z,9β,11α,13E)-17,17-propano-11,16-dihydroxy-9-chloro-20-norprost-5,13-dienoicacid.

(15) The endogenous repair factor production accelerator according tothe above (1), wherein the EP4 agonist is a compound represented byformula (I-b):

wherein R represents hydroxyl, C1-6 alkoxy or —NR^(6b)R^(7b);

R^(6b) and R^(7b) each independently represents hydrogen or C1-4 alkyl;

R^(2b) represents oxo, halogen or —O—COR^(8b);

R^(8b) represents C1-4 alkyl, phenyl or phenyl(C1-4 alkyl);

R^(3b) represents hydrogen or hydroxyl;

R^(4ab) and R^(4bb) each independently represents hydrogen or C1-4alkyl;

R^(5b) represents phenyl substituted with a group of the following i) toiv):

i) 1 to 3 of

C1-4 alkoxy-C1-4 alkyl,

C2-4 alkenyloxy-C1-4 alkyl,

C2-4 alkynyloxy-C1-4 alkyl,

C3-7 cycloalkyloxy-C1-4 alkyl,

C3-7 cycloalkyl(C1-4 alkoxy)-C1-4 alkyl,

phenyloxy-C1-4 alkyl,

phenyl-C1-4 alkoxy-C1-4 alkyl,

C1-4 alkylthio-C1-4 alkyl,

C2-4 alkenylthio-C1-4 alkyl,

C2-4 alkynylthio-C1-4 alkyl,

C3-7 cycloalkylthio-C1-4 alkyl,

C3-7 cycloalkyl(C1-4 alkylthio)-C1-4 alkyl,

phenylthio-C1-4 alkyl, or

phenyl-C1-4 alkylthio-C1-4 alkyl,

ii) C1-4 alkoxy-C1-4 alkyl and C1-4 alkyl,

C1-4 alkoxy-C1-4 alkyl and C1-4 alkoxy,

C1-4 alkoxy-C1-4 alkyl and hydroxy,

C1-4 alkoxy-C1-4 alkyl and halogen,

C1-4 alkylthio-C1-4 alkyl and C1-4 alkyl,

C1-4 alkylthio-C1-4 alkyl and C1-4 alkoxy,

C1-4 alkylthio-C1-4 alkyl and hydroxy, or

C1-4 alkylthio-C1-4 alkyl and halogen,

iii) haloalkyl or hydroxy-C1-4 alkyl, or

iv) C1-4 alkyl and hydroxy; and

represents a single bond or a double bond, and

wherein, when R^(2b) is —O—COR^(8b), the 8-9 position represents adouble bond,

a salt thereof or a cyclodextrin clathrate thereof

(16) The endogenous repair factor production accelerator according tothe above (15), wherein the EP4 agonist is

(1)(11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid, or

(2)(11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid methyl ester.

(17) A method for accelerating production of an endogenous repair factorin a mammal, which comprises administering to a mammal an effectiveamount of one or at least two selected from a PGI2 agonist, an EP2agonist and an EP4 agonist.

(18) A method for preventing and/or treating organ diseases in a mammal,which comprises administering to a mammal an effective amount of one orat least two selected from a PGI2 agonist, an EP2 agonist and an EP4agonist.

(19) Use of one or at least two selected from a PGI2 agonist, an EP2agonist and an EP4 agonist for preparing an endogenous repair factorproduction accelerator.

(20) Use of one or at least two selected from a PGI2 agonist, an EP2agonist and an EP4 agonist for preparing an agent for preventing and/ortreating organ diseases.

(21) A pharmaceutical composition which comprises the endogenous repairfactor production accelerator according to the above (1) in combinationwith one or at least two selected from an anti-thrombus agent, acirculation improving agent, a bronchial smooth muscle dilator, ananti-inflammatory drug, a local anesthetic, an analgesic, a bone cement,an joint lubricant, a PG derivative, an endogenous repair factorprotein, an endogenous repair factor gene and a stem cell.

In the specification, examples of the C1-4 alkyl include straight chainor branched chain C1-4 alkyl such as methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl and tert-butyl, and the like.

In the specification, examples of the C1-6 alkyl include straight chainor branched chain C1-6 alkyl such as methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, and thelike.

In the specification, examples of the C1-8 alkyl include straight chainor branched chain C1-8 alkyl such as methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl andoctyl, and the like.

In the specification, examples of the C2-8 alkenyl include straightchain or branched chain C2-8 alkenyl such as ethenyl, propenyl, butenyl,pentenyl, hexenyl, heptenyl and octenyl, and the like.

In the specification, examples of the C2-8 alkynyl include straightchain or branched chain C2-8 alkynyl such as ethynyl, propynyl, butynyl,pentynyl, hexynyl, heptynyl and octynyl, and the like.

In the specification, examples of the C1-4 alkoxy include straight chainor branched chain C1-4 alkoxy such as methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, isobutoxy, sec-butoxy and lei,-butoxy, and thelike.

In the specification, examples of the C1-6 alkoxy include straight chainor branched chain C1-6 alkoxy such as methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy andhexyloxy, and the like.

In the specification, examples of the C2-4 alkenyloxy include straightchain or branched chain C2-4 alkenyloxy such as ethenyloxy, propenyloxyand butenyloxy, and the like.

In the specification, examples of the C2-4 alkynyloxy include straightchain or branched chain C2-4 alkynyloxy such as ethynyloxy, propynyloxyand butynyloxy, and the like.

In the specification, examples of the C1-4 alkylthio include straightchain or branched chain C1-4 alkylthio such as methylthio, ethylthio,n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthioand tert-butylthio, and the like.

In the specification, examples of the C2-4 alkenylthio include straightchain or branched chain C2-4 alkenylthio such as ethenylthio,propenylthio and butenylthio, and the like.

In the specification, examples of the C2-4 alkynylthio include straightchain or branched chain C2-4 alkynylthio such as ethynylthio,propynylthio and butynylthio, and the like.

In the specification, examples of the halogen include fluorine,chlorine, bromine and iodine atoms and the like.

In the specification, examples of the trihalomethyl include methyltri-substituted with iodine atom, bromine atom, fluorine atom orchlorine atom.

In the specification, examples of the C4-7 cycloalkyl includecyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.

In the specification, examples of the C3-7 cycloalkyl includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and thelike.

In the specification, examples of the 4- to 7-membered monocyclic ringcontaining one nitrogen atom include azate, azole, pyridine and azepinrings or rings in which these rings are partially or entirely saturated.

In the specification, examples of the C3-7 cycloalkyloxy includecyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy,cycloheptyloxy and the like.

In the specification, examples of the C3-7 cycloalkylthio includecyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio,cycloheptylthio and the like.

In the specification, examples of the prodrug of the compoundrepresented by formula (I-a) include (1) a compound in which R^(a) isCOOR^(10a) (R^(10a) represents C1-6 alkyl), (2) a compound in whichR^(a) is CONR^(12a)R^(13a) (R^(12a) and R^(13a) each independentlyrepresents hydrogen or C1-6 alkyl), and (3) a compound in which R^(a) isCOOR^(10a) (R^(10a) has the same meaning as described above), R^(1a) isR^(11a)—COO (R^(11a) represents C1-4 alkyl, C1-4 alkoxy, phenyl,phenyl-C1-4 alkyl, R^(14a)—OOC—C1-4 alkyl or R^(14a)—OOC—C2-4 alkenyl(R^(14a) represents hydrogen or C1-4 alkyl), and the 8-9 position is adouble bond.

In the specification, examples of the endogenous repair factor includevascular endothelial growth factor (VEGF), hepatocyte growth factor(HGF), various fibroblast growth factors (a/b FGF), transformationgrowth factor-β (TGF-β), platelet derived growth factor (PDGF),angiopoietin, hypoxia inducing factor (HEF), insulin-like growth factor(IGF), bone morphogenetic protein (BMP), connective tissue growth factor(CTGF), epidermal growth factor (EGF) and the like, growth factors oftheir families and the like.

According to the description, for example, an IP agonist is alsoincluded in the PGI2 agonist.

According to the description, a prodrug is also included in the PGI2agonist, EP2 agonist or EP4 agonist. The prodrug is a compound which isconverted into its active form by through its reaction with an enzyme,gastric acid or the like in the living body. As the prodrug of the PGI2agonist, EP2 agonist or EP4 agonist, for example when the PGI2 agonist,EP2 agonist or EP4 agonist has amino group, examples include compoundsin which the amino group is acylated, alkylated or phosphorylated (e.g.,compounds in which the amino group of the PGI2 agonist, EP2 agonist orEP4 agonist is eicosanoylated, alanylated, pentylaminocarbonylated,(5-methyl-2-oxo-1,3-dioxolan-4-yl)methoxycarbonylated,tetrahydrofuranylated, pyrrolidylmethylated, pivaloyloxymethylated,acetoxymethylated or tert-butylated); when the PGI2 agonist, EP2 agonistor EP4 agonist has hydroxyl group, examples include compounds in whichthe hydroxyl group is acylated, alkylated, phosphorylated or borated(e.g., compounds in which the hydroxyl group of the PGI2 agonist, EP2agonist or EP4 agonist is acetylated, palmitoylated, propanoylated,pivaloylated, succinylated, fumarylated, alanylated ordimethylaminomethylcarbonylated); and when the PGI2 agonist, EP2 agonistor EP4 agonist has carboxyl, examples include compounds in which thecarboxyl is esterificated or amidated (e.g., compounds in which thecarboxyl of the PGI2 agonist, EP2 agonist or EP4 agonist is ethylesterificated, phenyl esterificated, carboxymethyl esterificated,dimethylaminomethyl esterificated, pivaloyloxymethyl esterificated,ethoxycarbonyloxyethyl esterificated, phthalidyl esterificated,(5-methyl-2-oxo-1,3-dioxolan-4-yl)methyl esterificated,cyclohexyloxycarbonylethyl esterificated or methylamidated). Thesecompounds can be produced by conventionally known methods. Also,prodrugs of the PGI2 agonist, EP2 agonist or EP4 agonist may be eitherhydrate or non-hydrate. In addition, prodrugs of the PGI2 agonist, EP2agonist or EP4 agonist may be those which are changed into the compoundsrepresented by formula (I) under physiological conditions, as describedin Iyakuhin-no Kaihatsu (Devlopment of Pharmaceutical Preparations),volume 7 “Bunshi Sekkei (Molecular Design)”, pp. 163-198, edited in 1990by Hirokawa Shoten.

Unless otherwise noted, all isomers are included in the presentinvention. For example, those of straight chain and branched chain areincluded in alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylene,alkenylene and alkynylene. In addition, all of the isomers regarding adouble bond, ring and condensed ring (E, Z, cis and trans isomers),isomers based on the presence of asymmetric carbon and the like (R and Sisomers, α and β configurations, enantiomers and diastereomers),optically active substances having optical activity (D, L, d and Iforms), polar forms by chromatographic separation (high polar form andlow polar form), symmetrical compounds, rotational isomers and theirmixtures having optional ratio and racemic mixtures are included in thepresent invention.

According to the present invention, unless otherwise noted and isevident to those skilled in the art,

means that it is bonded to the opposite side of the space (namely αconfiguration),

means that it is bonded to this side of the space (namely βconfiguration),

means that it is α configuration, β configuration or a mixture thereof,and

means the it is a mixture of α configuration and β configuration.

All of pharmacologically acceptable salts are included in the salts ofthe compounds represented by formula (I), formula (I-a) and formula(I-b). It is preferable that the pharmacologically acceptable salts donot have toxicity and are soluble in water. Examples of the suitablesalt of the compound represented by formula (I) include salts of alkalimetals (potassium, sodium, lithium, etc.), salts of alkaline earthmetals (calcium, magnesium, etc.), ammonium salts (tetramethylammoniumsalt, tetrabutylammonium salt, etc.), salts of organic amines(triethylamine, methylamine, dimethylamine, cyclopentylamine,benzylamine, phenethylmine, piperidine, monoethanolamine,diethanolamine, tris(hydroxymethyl)methylamine, lysine, arginine,N-methyl-D-glucamine, etc.), and acid addition salts (inorganic acidsalts (hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate,nitrate, etc.), organic acid salts (acetate, trifluoroacetate, lactate,tartarate, oxalate, fumarate, maleate, benzoate, citrate,methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate,isethionate, glucuronate, gluconate, etc.) and the like). Also includedin the salts of the compounds of the present invention are solvates, orsolvates of alkali (alkaline earth) metal salts, ammonium salts, organicamine salts and acid addition salts of the compounds of the presentinvention. It is preferable that the solvates are nontoxic and solublein water. Examples of appropriate solvate include solvates of water,alcohol system solvents (ethanol, etc.) and the like.

The compounds of the present invention are converted intopharmacologically acceptable salts by conventionally known methods.

In addition, quaternary ammonium salt is also included in the salts. Thequaternary ammonium salt means that the nitrogen atom of the compoundrepresented by formula (I) is quaternarized by R⁰ (R⁰ represents C1-8alkyl, or C1-8 alkyl substituted by phenyl).

Also, N-oxide is included in the salts. The compounds of the presentinvention can be made N-oxide by optional method. The N-oxide means thatthe nitrogen atom of the compound represented by formula (I) isoxidized.

The compounds represented by formulae (I-a) and (I-b) can be convertedinto cyclodextrin clathrate by the method described in the specificationof JP-B-50-3362, JP-B-52-31404 or JP-B-61-52146 using α-, β- orγ-cyclodextrin or a mixture thereof Since stability is improved andwater-solubility is increased by their conversion into cyclodextrinclathrate, it is convenient when they are used as pharmaceuticalpreparations.

As a preferable embodiment according to this description, a preventiveand/or therapeutic agent for (ischemic) organ diseases by a persistentpreparation of a prostaglandin (PG) I2 agonist, an EP2 agonist or an EP4agonist is preferable, and more preferred is a preventive and/ortherapeutic agent for (ischemic) organ diseases by topicaladministration of a persistent preparation of a prostaglandin (PG) I2agonist, an EP2 agonist or an EP4 agonist to an ischemic region or aninjured region.

All of the PGI2 agonists so far known and PGI2 agonists which will bediscovered in the future are included in the PGI2 agonist of thedescription.

For example, the compound represented by formula (I) or a salt thereofis preferable as the PGI2 agonist.

In addition, in formula (I), preferred as

In formula (I), preferred as R² is (iii) phenyl or C4-7 cycloalkyl, (iv)4- to 7-membered monocyclic ring containing one nitrogen atom, (v) C1-C4alkyl substituted with a benzene ring or C4-7 cycloalkyl, or (vi) C1-4alkyl substituted with 4- to 7-membered monocyclic ring containing onenitrogen atom, and particularly preferred is (iii) phenyl or C4-7cycloalkyl or (iv) 4- to 7-membered monocyclic ring containing onenitrogen atom.

In formula (I), preferred as R³ is (ii) phenyl or C4-7 cycloalkyl, (iii)4- to 7-membered monocyclic ring containing one nitrogen atom, (iv) C1-4alkyl substituted with a benzene ring or C4-7 cycloalkyl, or (v) C1-4alkyl substituted with 4- to 7-membered monocyclic ring containing onenitrogen atom, and particularly preferred is (ii) phenyl or C4-7cycloalkyl or (iii) 4- to 7-membered monocyclic ring containing onenitrogen atom. In addition, more preferred is

Compound 1:

(E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid:

orCompound 2:(Z)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid:

In addition, examples of the other PGI2 agonist include beraprost sodium((±)-(1R,2R,3 aS, 8bS)-2,3,3 a, 8b-terahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butanoicacid sodium salt), OP-2507 (5-{(3aR,4R,6aS)-5-hydroxy-4-[(1E,3S)-3-hydroxy-3-(cis-4-propylcyclohexyl)prop-1-enyl-3,3a,4,5,6,6a-hexahydrocyclopenta[b]pyrrol-2-yl}pentanoic acid methylester), OP-41483((5E)-5-[(3aS,4R,5R,6aS)-4-[(1E,3S)-3-cyclopentyl-3-hydroxyprop-1-enyl]-5-hydroxyhexahydropentalene-2(1H)-ylidene]pentanoicacid) and the like, and a chemically stable carbacyclin system PGI2relate compound is preferable.

All of the EP2 agonists so far known and EP2 agonists which will bediscovered in the future are included in the EP2 agonist of thedescription. For example, as the EP2 agonists so far known, thecompounds described in JP-A-11-193268, namely the above-describedcompounds represented by formula (I-a), salts thereof, prodrugs thereofor cyclodextrin clathrate thereof can be cited.

Examples of the other EP2 agonist include compounds described in thespecifications of WO99/33794, EP-A-974580, WO95/19964, WO98/28264,WO99/19300, EP-A-0911321, WO98/58911, U.S. Pat. No. 5,698,598, U.S. Pat.No. 6,376,533, U.S. Pat. No. 4,132,738, U.S. Pat. No. 3,965,143 and thelike.

Among the compounds represented by formula (I-a), Compound 3:(5Z,9β,11α,13E)-17,17-propano-11,16-dihydroxy-9-chloro-20-norprost-5,13-dienoicacid:

(the compound described in Example 17 of the specification ofJP-A-11-193268), a lysine salt thereof or an α-cyclodextrin clathratethereof, and the like can exemplified as more preferable compounds.

All of the EP4 agonists so far known and EP4 agonists which will bediscovered in the future are included in the EP4 agonist of thedescription. For example, as the EP4 agonists so far known, thecompounds described in the specification of WO00/03980, namely theabove-described compounds represented by formula (I-b), salts thereof orcyclodextrin clathrate thereof can be cited.

Examples of the other EP4 agonist include compounds described in thespecifications of WO99/02164, WO00/16760, WO00/18744, WO00/21542,WO00/38663, WO00/38690, WO00/38667, WO00/40248, WO00/54808, WO00/54809,WO01/10426, EP-A-1110949, EP-A-1121939, EP-A-1132086, WO200172268,JP-A-2002-104939, WO02/42268, JP-A-2002-179595, WO02/47669, WO02/64564,WO03/035064, WO03/053923, , U.S. Pat. No. 6,552,067 and the like.

Among the compounds represented by formula (I-b), Compound 4:(11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid:

(the compound described in Example 3 of the specification ofWO00/03980),(11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid methyl ester and the like can exemplified as more preferablecompounds.Production Methods of Compounds According to the Present Invention:

Among the PGI2 agonists to be used in the present invention, forexample, production methods of the compounds represented by formula (I)are disclosed in the specification of JP-A-6-87811.

For example,(E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid (Compound 1) is described in Example 2(g).

Also,(Z)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid (Compound 2) is described in Example 2(f).

Production method of Beraprost((±)-(1R,2R,3aS,8bS)-2,3,3a,8b-terahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyl-l-octen-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butanoic acid sodium salt)is disclosed in the specification of JP-A-62-134787.

Production method of OP-2507(5-{(3aR,4R,6aS)-5-hydroxy-4-[(1E,3S)-3-hydroxy-3-(cis-4-propylcyclohexyl)prop-1-enyl-3,3a,4,5,6,6a-hexahydrocyclopenta[b]pyrrol-2-yl}pentanoicacid methyl ester) is disclosed in the specification of JP-A-61-30519.

Production method of OP-41483((5E)-5-[(3aS,4R,5R,6aS)-4-[(1E,3S)-3-cyclopentyl-3-hydroxyprop-1-enyl]-5-hydroxyhexahydropentalene-2(1H)-ylidene]pentanoicacid) is disclosed in the specification of JP-A-54-130543.

Production methods of the compounds represented by formula (I-a) aredisclosed in the specification of JP-A-11-193268. For example,(5Z,9β,11α,13E)-17,17-propano-11,16-dihydroxy-9-chloro-20-norprost-5,13-dienoicacid (Compound 3) is described in Example 17.

Production methods of the compounds represented by formula (I-b) aredisclosed in the specification of WO00/03980. For example,(11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid (Compound 4) is described in Example 3.

Toxicity:

Since toxicity of the agents of the present invention is very low, theyare sufficiently safe for using as medicine.

INDUSTRIAL APPLICABILITY

Application to Pharmaceutical Preparations:

Since the PGI2 agonist, EP2 agonist or EP4 agonist has the angiogenesisaccelerating action, it is useful as preventive and/or therapeuticagents for ischemic organ diseases (e.g., arteriosclerosis obliterans,Buerger disease, Raynaud disease, cardiovascular diseases (e.g.,myocardial infarction, angina pectoris, etc.), diabetic neuropathy,spinal canal stenosis, ischemic brain disease (e.g., cerebrovascularaccidents, cerebral infarction, etc.), pulmonary hypertension, bonefracture, Alzheimer disease, etc.). In addition, since the PGI2 agonist,EP2 agonist or EP4 agonist has the endogenous repair factor productionaccelerating action, based on its action to induce differentiation fromrespective stem cells for repairing tissues, it is useful as preventiveand/or therapeutic agents for various organ diseases (for example, liverdiseases (e.g., fluminant hepatitis, acute hepatitis, hepatic cirrhosis,fatty liver, liver transplantation, etc.), kidney diseases (e.g., acuterenal insufficiency, chronic renal insufficiency, etc.), lung diseases(e.g., acute pneumonia, pulmonary fibrosis, pulmonary hypertension,chronic obstructive pulmonary disease (COPD), asthma, etc.), pancreasdiseases (e.g., diabetes mellitus, chronic pancreatitis, etc.), bonediseases (e.g., osteoarthritis, articular rheumatism, osteoporosis, bonefracture, periosteunm injury, etc.), digestive organ diseases (e.g.,gastric ulcer, duodenal ulcer, ulcerative colitis, Crolin disease,etc.), nerve degeneration diseases (e.g., stroke, Parkinson disease,Alzheimer disease, spinal canal stenosis, cerebrovascular accidents,moyamoya disease, etc.), diabetic complications (e.g., nerve disorder,skin ulcer, nephropathy, retinal disease, etc.), vascular endothelialcell diseases (e.g., restenosis after PTCA (percutaneous transluminalcoronary angiopathy), arteriosclerosis, etc.), heart diseases (e.g.,supraventricular tachyarrhythmia, congestive heart failure, coronaryartery disease, sudden cardiomyopathy, dilated cardiomyopathy, etc.),dental diseases (e.g., periodontal disease, tooth extraction wound, oralwound, periodontal tissue disease, etc.), decubitus, glaucoma, alopecia,and the like).

As the active ingredient of the endogenous repair factor productionaccelerator of the present invention, one or two or more speciesoptionally selected from the homologous group and heterogeneous group ofPGI2 agonists, EP2 agonists and EP4 agonists may be used at acombination of optional ratio.

For example, PGE1, PGE2 and PGI2, derivatives thereof (e.g., 6-oxoPGE1,ornoprostil, limaprostil, enprostil, misoprostol, etc.), prodrugsthereof, persistent preparations (sustained release preparations)thereof (e.g., lipoPGE1, etc.) and endogenous inducers thereof are alsoincluded in the PGI2 agonist, EP2 agonist or EP4 agonist of the presentinvention, and one or two or more of them may be optionally blended andused.

The agent of the present invention may be administered as a concomitantdrug in combination with other agent for (1) complementing and/orreinforcing the preventive and/or therapeutic effect of the agent of thepresent invention, (2) improving kinetics and absorption of the agent ofthe present invention and reducing its dose, and/or (3) alleviating sideeffects of the agent of the present invention.

The concomitant drug of the agent of the present invention with otheragent may be administered in the form of a combination drug in whichboth components are formulated in one pharmaceutical preparation, oradministered as separate pharmaceutical preparations. When administeredas the separate pharmaceutical preparations, it includes simultaneousadministration and differential time administration. In addition, thedifferential time administration may be carried out by firstlyadministering the agent of the present invention and then administeringthe other agent, or by firstly administering the other agent and thenadministering the agent of the present invention, and the respectiveadministration methods mat be the same or different from each other.

The other agent may be a low molecular compound, a high molecularprotein, polypeptide, polynucleotide (DNA, RNA or a gene), antisense,decoy or antibody, or a vaccine or a stem cell or the like separatedfrom a tissue. Dose of the other agent can be optionally selected basedon the clinically used dose. In addition, blending ratio of the agent ofthe present invention with the other agent can be optionally selecteddepending on the age and body weight of the subject to be administered,administration method, administration period, disease to be treated,symptoms, combination and the like. For example, from 0.01 to 100 partsby mass of the other agent may be used based on 1 part by mass of theagent of the present invention. The other agent may be administered asan optional ratio of combination of one or two or more speciesoptionally selected from the homologous group and heterogeneous groupshown in the following.

The disease in which its preventive and/or therapeutic effect is exertedby the above-described concomitant drug is not particularly limited, andit may be any disease in which the preventive and/or therapeutic effectof the agent of the present invention can be complemented and/orreinforced.

Examples of the other agent include an anti-thrombus agent, acirculation improving agent, a bronchial smooth muscle dilator, ananti-inflammatory drug, a local anesthetic, an analgesic, a bone cement,an joint lubricant, a prostaglandin derivative, an endogenous repairfactor protein, an endogenous repair factor gene, various organ stemcells and the like.

Examples of the anti-thrombus agent include heparin preparations(heparin sodium, heparin calcium, dalteparin sodium, etc.), oralanticoagulants (warfarin potassium, etc.), antithrombin agents (gabexatemesylate, nafamostat mesylate, argatroban, etc.), anti-plateletagglutination inhibitors (aspirin, dipyridamole, ticlopidinehydrochloride, beraprost sodium, cilostazol, ozagrel sodium,sarpogrelate hydrochloride, ethyl eicosapentanoate, etc.), thrombolyticagents (urokinase, tisokinase, alteplase, nateplase, monteplase,pamiteplase, etc.), factor Xa inhibitors, factor VIla inhibitors and thelike.

Examples of the circulation improving agent include ifenprodiltartarate, aniracetam, donepezil hydrochloride, amantadinehydrochloride, nicergoline, ibudilast, a papaverine system, a nicotinesystem, a calcium antagonist, a β receptor agonist, an α receptorantagonist and the like.

Examples of the bronchial smooth muscle dilator include β 2 stimulants(e.g., ephedrine hydrochloride, isiprenali sulfate, salbutamol sulfate,tulobuterol hydrochloride, etc.), theophylline drugs (e.g.,diprophylline, aminophylline, choline theophylline, etc.), oranti-choline drugs (e.g., ipratropium bromide, flutropium bromide,oxytropium bromide, etc.).

Examples of the local anesthetic include a steroid preparation,procaine, cocaine hydrochloride, lidocaine hydrochloride, ropivacainehydrochloride and the like.

Examples of the an analgesic include non-steroidal anti-inflammatorydrugs (NSAID) such as aspirin, indometacin, diclofenac, meloxicam andcelecoxib, opioid analgesic such as codein and morphine, pentazocine,buprenorphine hydrochloride, eptazocine hydrobromide and the like.

Examples of the bone cement include calcium phosphate and the like.

Examples of the joint lubricant include suvenyl and the like.

Examples of the prostaglandin derivative include PGE1, PGE2 and PGI2 orprodrugs thereof, lipoPGE1, 6-oxoPGE1, 6-oxoPGE1 derivatives,ornoprostil, limaprostil, enptostil, misoprostol and the like.

Examples of the endogenous repair factor according to the “endogenousrepair factor protein and endogenous repair factor gene” includevascular endothelial growth factor (VEGF), hepatocyte growth factor(HGF), various fibroblast growth factors (a/b FGF), transformationgrowth factor-β (TGF-β), platelet derived growth factor (PDGF),angiopoietin, hypoxia inducing factor (HIF), insulin-like growth factor(IGF), bone morphogenetic protein (BMP), connective tissue growth factor(CTGF) and epidermal growth factor (EGF), growth factors of theirfamilies and the like.

In addition, not only the compounds so far found but also those whichwill be discovered in the future are also included in the other agentscapable of complementing and/or reinforcing the preventive and/ortherapeutic agents of the present invention, based on theabove-described mechanism.

When the agent of the present invention, or the concomitant drug of theagent of the present invention with other agent, is used for theabove-described purpose, it is generally administered systemically ortopically in the oral or parenteral form.

Its dose varies depending on the age, body weight, symptoms, therapeuticeffect, administration method, treating period and the like, but isusually within the range of from 1 ng to 100 mg per adult per once, fromonce to several times a day by oral administration, or within the rangeof from 0.1 ng to 50 mg per adult per once, from once to several times aday, from once to several times a week, or from once to several times in3 months by parenteral administration in the form of a persistentpreparation, or continuously administered into a vein within the rangeof from 1 hour to 24 hours a day. Since the dose varies under variousconditions as a matter of course as described above, there is a case inwhich a smaller dose than the above range is sufficient or a case whichrequires the administration exceeding the range.

When the agent of the present invention, or the concomitant drug of theagent of the present invention with other agent, is administered, it isused as solid preparations for internal use or liquid preparations forinternal use for oral administration, or as injections, subcutaneous orintramuscular injections, external preparations, suppositories, eyedrops, inhalations, medical device-containing preparations and the likefor parental administration.

The solid preparation for internal use for use in the oraladministration includes tablets, pills, capsules, powders, granules andthe like. Hard capsules and soft capsules are included in the capsules.

In such a solid preparation for internal use, one or more activesubstances are used as such, or mixed with a filler (lactose, mannitol,glucose, microcrystalline cellulose, starch, etc.), a binder(hydroxypropylcellulose, polyvinyl pyrrolidone, magnesiumaluminometasilicate, etc.), a disintegerating agent (calcium celluloseglycolate, etc.), a lubricant (magnesium stearate, etc.), a stabilizingagent, a solubilization assisting agent (glutamic acid, aspartic acid,etc.) and the like and used by making the mixture into a pharmaceuticalpreparation. If necessary, this may be coated with a coating agent(sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulosephthalate, etc.), or coated with two or more layers. Further capsules ofan absorbable substance such as gelatin are included.

The liquid preparation for internal use for use in the oraladministration includes pharmaceutically acceptable solutions,suspensions, emulsions, syrups, elixirs and the like. In such a liquidpreparation, one or more active ingredient are dissolved, suspended oremulsified in a generally used diluent (purified water, ethanol, a mixedsolution thereof, etc.). In addition, this liquid preparation maycontain a moistening agent, a suspending agent, an emulsifying agent, asweetener, a flavor, an aromatic, a preservative, a buffer and the like.

Dosage forms for external use for use in parenteral administrationinclude, for example, ointments, gels, creams, fomentations, adhesivepreparations, liniments, sprays, inhalations, sprays, aerosols, eyedrops, nasal drops and the like. In addition, these may be sealed with abiodegradable polymer and used as medical devices (surgical suture, abolt for use in bone fracture treatment, etc.). They contain one or moreactive ingredient and are prepared by a conventionally known method orbased on a generally used formula.

The ointments are produced by a conventionally known method or based ona generally used formula. For example, they are prepared by suspendingor melting one or more active ingredients in a base. The ointment baseis selected from those which are conventionally known or generally used.For example, a single substance or a mixture of two or more substancesare used, which are selected from higher fatty acids or higher fattyacid esters (adipic acid, myristic a cid, palmitic acid, stearic acid,oleic acid, adipic acid ester, myristic acid ester, palmitic acid ester,stearic acid ester, oleic acid ester, etc.), waxes (beeswax, whale wax,ceresine, etc.), surfactants (polyoxyethylene alkyl ether phosphoricacid ester, etc.), higher alcohols (cetanol, stearyl alcohol,cetostearyl alcohol, etc.), silicon oil (dimethyl polysiloxane, etc.),hydrocarbons (hydrophilic vaseline, white vaseline, purified lanoline,liquid paraffin, etc.), glycols (ethylene glycol, diethylene glycol,propylene glycol, polyethylene glycol, macrogol, etc.), plant oil(castor oil, olive oil, sesame oil, turpentine oil, etc.), animal oil(mink oil, egg yolk oil, squalane, squalene, etc.), water, absorptionaccelerators and rash preventing agents. In addition, moisture keepingagents, preservatives, stabilizers, antioxidants, flavors and the likemay be contained.

The gels are produced by a conventionally known method or based on agenerally used formula. For example, they are prepared by melting one ormore active ingredients in a base. The gel base is selected from thosewhich are conventionally known or generally used. For example, a singlesubstance or a mixture of two or more substances are used, which areselected from lower alcohols (ethanol, isopropyl alcohol, etc.), gellingagents (carboxymethyl cellulose, hydroxyethyl cellulose,hydroxypropylcellulose, ethyl cellulose, etc.), neutralizing agents(triethanolamine, diisopropanolamine, etc.), surfactants (polyethyleneglycol monostearate, etc.), gums, water, absorption accelerators andrash preventing agents. In addition, preservatives, antioxidants,flavors and the like may be contained.

The creams are produced by a conventionally known method or based on agenerally used formula. For example, they are prepared by melting oremulsifying one or more active ingredients in a base. The cream base isselected from those which are conventionally known or generally used.For example, a single substance or a mixture of two or more substancesare used, which are selected from higher fatty acid esters, loweralcohols, hydrocarbons, polyhydric alcohols (propylene glycol,1,3-butylene glycol, etc.), higher alcohols (2-hexyldecanol, cetanol,etc.), emulsifiers (polyoxyethylene alkyl ethers, fatty acid esters,etc.), water, absorption accelerators and rash preventing agents. Inaddition, preservatives, antioxidants, flavors and the like may becontained.

The fomentations are produced by a conventionally known method or basedon a generally used formula. For example, they are prepared by meltingone or more active ingredients in a base and then spreading and coatingthe resulting kneaded material on a support. The fomentation base isselected from those which are conventionally known or generally used.For example, a single substance or a mixture of two or more substancesare used, which are selected from thickeners (polyacrylic acid,polyvinyl pyrrolidone, acacia, starch, gelatin, methyl cellulose, etc.),moistening agents (urea, glycerol, propylene glycol, etc.), fillers(kaolin, zinc oxide, talc, calcium, magnesium, etc.), water,solubilizing agents, tackifiers and rash preventing agents. In addition,preservatives, antioxidants, flavors and the like may be contained.

The adhesive preparations are produced by a conventionally known methodor based on a generally used formula. For example, they are prepared bymelting one or more active ingredients in a base and then spreading andcoating the resulting material on a support. The adhesive preparationbase is selected from those which are conventionally known or generallyused. For example, a single substance or a mixture of two or moresubstances are used, which are selected from polymer bases, oils andfats, higher fatty acids, tackifiers and rash preventing agents. Inaddition, preservatives, antioxidants, flavors and the like may becontained.

The liniments are produced by a conventionally known method or based ona generally used formula. For example, they are prepared by dissolving,suspending or emulsifying one or more active ingredients in a singlesubstance or two or more substances selected from water, alcohols(ethanol, polyethylene glycol, etc.), higher fatty acids, glycerol,soap, emulsifiers, suspending agents and the like. In addition,preservatives, antioxidants, flavors and the like may be contained.

In addition to the generally used diluents, the sprays and inhalationsmay contain stabilizers such as sodium hydrogen sulfite and bufferagents capable of giving tonicity, for example, tonicity agents such assodium chloride, sodium citrate and citric acid. Production methods ofsprays are illustratively described in, for example, U.S. Pat. No.2,868,691 and U.S. Pat. No. 3,095,355.

Solutions, suspensions, emulsions and solid injections which are used bydissolving or suspending in a solvent prior to use are included in theinjections for parenteral administration. The injections are used bydissolving, suspending or emulsifying one or more active ingredients ina solvent. These injections may be injected into a vein, an artery,muscle, under the skin, into the brain, a joint, a bone and othertopical regions of organs, or directly administered using aneedle-equipped blood vessel catheter or the like. As the solvent, forexample, distilled water for injection, physiological saline, plant oil,alcohols such as propylene glycol, polyethylene glycol and ethanol, andcombinations thereof are used. In addition, such injections may containa stabilizer, a solubilization assisting agent (glutamic acid, asparticacid, Polysorbate 80 (registered trade mark), etc.), a suspending agent,an emulsifying agent, a soothing agent, a buffer agent, a preservativeand the like. These are produced by sterilizing at the final step or byan aseptic operation. In addition, it is possible to prepare an asepticsolid preparation, such as a freeze-dried preparation, and use it bydissolving in sterilized or aseptic distilled water for injection orother solvent prior to its use.

The eye drops for parenteral administration include an eye drop liquid,a suspension type eye drop liquid, an emulsion type eye drop liquid, aprior to use dissolution type eye drop liquid and an eye ointment.

These eye drops are produced in accordance with a conventionally knownmethod. For example, they are used by dissolving, suspending oremulsifying one or more active ingredient in a solvent. As the solventof eye drops, for example, sterilized purified water, physiologicalsaline, other aqueous solvent or non-aqueous solvent for injection(e.g., plant oil, etc.) and the like and combinations thereof are used.If necessary, the eye drops may further contain substances optionallyselected from tonicity agents (sodium chloride, concentrated glycerol,etc.), buffer agents (sodium phosphate, sodium acetate, etc.),surfactants (Polysorbate 80 (registered trade mark), polyoxyl 40stearate, polyoxyethylene hardened castor oil, etc.), stabilizers(sodium citrate, sodium edetate, etc.), antiseptics (benzalkoniumchloride, paraben, etc.) and the like. These are produced by sterilizingat the final step or by an aseptic operation. In addition, it ispossible to prepare an aseptic solid preparation, such as a freeze-driedpreparation, and use it by dissolving in sterilized or aseptic distilledpurified water or other solvent prior to its use.

As the inhalations for parenteral administration, aerosols, powders forinhalation or solutions for inhalation are included, and said solutionsfor inhalation may be in a form which is used by dissolving orsuspending in water or other appropriate solvent prior to its use.

These inhalations are produced in accordance with conventionally knownmethods.

For example, in the case of solutions for inhalation, they are preparedby optionally selecting antiseptics (benzalkonium chloride, paraben,etc.), coloring agents, buffer agents (sodium phosphate, sodium acetate,etc.), tonicity agents (sodium chloride, concentrated glycerol, etc.),thickeners (carboxyvinyl polymer, etc.), absorption accelerators and thelike, if necessary.

In the case of powders for inhalation, they are prepared by optionallyselecting lubricants (stearic acid and a salt thereof, etc.), binders(starch, dextrin, etc.), fillers (lactose, cellulose, etc.), coloringagents, antiseptics (benzalkonium chloride, paraben, etc.), absorptionaccelerators and the like, if necessary.

When solutions for inhalation are administered, a sprayer (atomizer ornebulizer) is generally used, and an inhalation administering device forpowders is generally used when powders for inhalation are used.

As other compositions for parenteral administration, suppositories forrectal administration, pessaries for vaginal administration and the likeare included, which contain one or more active ingredients and areformulated in the usual way.

Application to Topical Regions:

As the topical administration of the present invention, theadministration method is not particularly limited, so long as the agentof the present invention or a concomitant drug of the agent of thepresent invention with other agent can be topically supplied to regionsof a disease. Its examples include injections and embedding agents to beused in muscle, under the skin, and in the skin, blood vessel, heartmuscle, alveoli, joint part, vertebra, bone part, tooth root part,injured organ and the like, medical device-containing preparations inwhich the agent of the present invention or a concomitant drug of theagent of the present invention with other agent is contained in amedical device (stent, fixing bolt, fixer, thread, etc.), or coatingagents coated with the same, solid preparations such as granules andpowders, adhesive preparations, gels, ointments, films, preparationenclosed in a biodegradable polymer, or enclosed medical devices and thelike.

As the persistent preparation of the present invention, thepharmaceutical preparation is not limited, so long as the activeingredient can be persistently supplied to a region of a disease. Itsexamples include sustained release injections (e.g., microcapsulepreparations, microsphere preparations, nanosphere preparations, etc.),embedding preparations (e.g., film preparations, etc.), ointments,coatings in which the active ingredient is contained or coated in amedical device (stent, fixing bolt, fixer, suture, etc.).

The microcapsule preparations, microsphere preparations and nanospherepreparations of the present invention are fine particle pharmaceuticalcomposition with a biodegradable polymer, which contains an activeingredient as the active ingredient.

A bioabsorbable polymer is present in the sustained drug release systemof the present invention, which is achieved by a natural polymer or asynthetic polymer. The mechanism for controlling the rate of sustainedrelease therefrom includes a degradation controlling type, a dispersioncontrolling type, a membrane permeation controlling type or the like.

Examples of the natural polymer as the bioabsorbable polymer of thepresent invention include plant-produced polysaccharides (e.g.,cellulose, starch, alginic acid, etc.), animal-produced polysaccharidesand proteins (e.g., chitin, chitosan, collagen, gelatin, albumin,glycosaminoglycan, etc.) and microorganism-produced polyesters andpolysaccharides (e.g., poly-3-hydroxyalkanoate, hyaluronic acid, etc.).

Also, examples of the biodegradable polymer include fatty acid esterpolymers or copolymers thereof, polyacrylic acid esters,polyhydroxybutyric acids, polyalkylene oxalates, polyortho esters,polycarbonate and polyamino acids, which can be used alone or as amixture thereof Examples of the fatty acid ester polymers or copolymersthereof include polylactic acid, polyglycolic acid, polycitric acid,polymalic. acid, polyethylene succinate, polybutylene succinate,poly-ε-caprolactone, polybutylene terephthalate adipate or lacticacid-glycolic acid copolymer, which can be used alone or as a mixturethereof In addition to these, poly α-cyano acrylic acid ester,poly-β-hydroxy butyric acid, polytrimethylene oxate, polyortho ester,polyortho carbonate, polyethylene carbonate, poly-γ-benzyl-L-glutamicacid, polyvinyl alcohol, polyester carbonate, polyacid anhydride,polycyano acrylate, polyphosphazine or poly-L-alanine can be used aloneor as a mixture thereof Preferred is polylactic acid, polyglycolic acidor a lactic acid-glycolic acid copolymer, and more preferred is a lacticacid-glycolic acid copolymer.

Average molecular weight of these biodegradable high molecular weightpolymers to be used in the present invention is preferably from about2,000 to about 800,000, more preferably from about 5,000 to about200,000. For example, in the case of polylactic acid, its weight averagemolecular weight is preferably from about 5,000 to about 100,000. It ismore preferably from about 6,000 to about 50,000. Polylactic acid can besynthesized in accordance with the conventionally known productionmethod. In the case of the lactic acid-glycolic acid copolymer, itscompositional ratio of lactic acid and glycolic acid preferably fromabout 100/0 to about 50/50 (w/w), particularly preferably from about90/10 to about 50/50 (w/w). Weight average molecular weight of thelactic acid-glycolic acid copolymer is preferably from about 5,000 toabout 100,000. It is more preferably from about 10,000 to about 80,000.The lactic acid-glycolic acid copolymer can be synthesized in accordancewith the conventionally known production method. In addition, in orderto control initial burst, basic amino acids (e.g., alginic acid, etc.)may be added.

According to the description, the weight average molecular weight is apolystyrene-based molecular weight measured by a gel permeationchromatography (GPC).

The above-described biodegradable high molecular weight polymer can bechanged depending on the strength of pharmacological activity of theactive ingredient and the drug release of interest, so long as theobject of the present invention is attained, and for example, it is usedin an amount of from about 0.2 to about 10,000 times (weight ratio),preferably from about 1 to about 1,000 times (weight ratio), more fromabout 1 to about 100 times (weight ratio), based on said physiologicallyactive substance.

The microspheres, microcapsules and nanocapsules of the presentinvention can be produced for example by a submerged drying method(e.g., o/w method, w/o method, w/o/w method, etc.), a phase separationmethod, a spray drying method, a supercritical fluid granulation methodor a method corresponding thereto.

Specific production methods on the submerged drying (o/w method) andspray drying are described in the following.

(1) In the submerged drying method (o/w method), an organic solventsolution of a biodegradable polymer is firstly prepared. It ispreferable that the organic solvent to be used in producing themicrospheres, microcapsules and nanocapsules of the present inventionhas a boiling point of 120° C. or less. Examples of the organic solventinclude halogenated hydrocarbons (e.g., dichloromethane, chloroform,etc.), aliphatic esters (e.g., ethyl acetate, etc.), ethers, aromatichydrocarbons, ketones (acetone, etc.) and the like. Two or more of themmay be used by mixing at an optional ratio. Desirable organic solvent isdichloromethane or acetonitrile. The organic solvent is preferablydichloromethane. Concentration of the biodegradable polymer in theorganic solvent solution varies depending on the molecular weight of thebiodegradable polymer, kind of the organic solvent and the like, but isgenerally selected within the range of from about 0.01 to about 80%(v/w). It is preferably from about 0.1 to about 70% (v/w), morepreferably from about 1 to about 60% (v/w).

An active ingredient is added to and dissolved in the thus obtainedorganic solvent solution of biodegradable polymer. Amount of this activeingredient to be added varies depending on the kind of agent,angiogenesis action, effect-persisting period of time and the like, butis from about 0.001% to about 90% (w/w), preferably from about 0.01% toabout 80% (w/w), more preferably from about 0.3% to 30% (w/w), asconcentration of the biodegradable high molecular polymer in the organicsolvent solution.

Next, the thus prepared organic solvent solution is further added to awater phase to form an o/w emulsion using a stirrer, emulsifier or thelike. The water phase volume in this case is selected generally fromabout 1 time to about 10,000 times of the oil phase volume. This isselected more preferably from about 2 times to about 5,000 times. Thisis selected particularly preferably from about 5 times to about 2,000times. An emulsifying agent may be added to the water phase of theabove-described outer phase. The emulsifying agent may be any agentwhich can generally form a stable o/w emulsion. Examples of theemulsifying agent include an anionic surfactant, a nonionic surfactant,a polyoxyethylene castor oil derivative, polyvinyl pyrrolidone,polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin and thelike. These may be used in an optional combination. Concentration of theemulsifying agent in the outer water phase is preferably from about0.001% to about 20% (w/w). It is more preferably from about 0.01% toabout 10% (w/w), particularly preferably from about 0.05% to about 5%(w/w).

A generally used method is employed for the evaporation of solvent inthe oil phase. The method is carried out under ordinary pressure orgradually reducing the pressure while stirring using a stirrer, magneticstirrer or the like, or using a rotary evaporator or the like whileadjusting the degree of vacuum. After fractionating the thus obtainedmicrospheres by centrifugation or filtration, the free activeingredient, emulsifying agent and the like adhered to the microspheresurface are washed several times repeatedly with, for example, asurfactant solution, an alcohol or the like, and the resultingmicrospheres are again dispersed in distilled water or a dispersionmedium containing a filler (mannitol, sorbitol, lactose, etc.) or thelike and freeze-dried. As the above-described o/w method, microspheresmay be produced by a method in which an active ingredient is dispersedin an organic solvent solution of biodegradable polymer, namely an s/o/wmethod.

(2) When microspheres are produced by a spray drying method, an organicsolvent in which a biodegradable polymer and an active ingredient aredissolved, or an emulsion of the same, is sprayed into a drying chamberof a spray dryer device (spray dryer) using a nozzle, and the organicsolvent or water in the fine particle droplets is evaporated within aextremely short period of time to prepare microspheres. As the nozzle,there are a double fluid nozzle type, a pressure nozzle type, a rotarydisc type and the like. In this case, for the purpose of preventingaggregation of microspheres, if necessary, it is effective to spray anorganic solvent or aqueous solution of an aggregation preventing agent(mannitol, lactose, gelatin, etc.) through another nozzle,simultaneously with the spraying of o/w emulsion. As the thus obtainedmicrospheres, more complete removal of water and solvent in themicrospheres is carried out under a reduced pressure, with heating, ifnecessary.

Examples of the film preparations include those in which theabove-described biodegradable polymer and active ingredient aredissolved in an organic solvent and then evaporated into dryness to forma film or in which the biodegradable polymer and active ingredient aredissolved in an appropriate solvent and then gelatinized by adding agranulation agent (celluloses, polycarbonates, etc.).

The microspheres, microcapsules and nanospheres of the present inventioncan be made into various dosage forms of pharmaceutical preparation, forexample, as such , or using a spherical, rod, needle, bolt, filamentous,pellet, film or cream-shaped pharmaceutical composition as the materialsubstance.

In addition, using this pharmaceutical preparation, it can beadministered as parenteral preparations for topical administration(e.g., injections and embedding agents to be used in muscle, under theskin, and in the skin, heart muscle, abdominal cavity, bronchus, bloodvessel, alveoli, injured region of vascular endothelium, brain, marrow,inside of dura mater, outside of dura mater, joint part, vertebra, bonepart, periodontal part, various organs and the like, solid preparationssuch as granules and powders, liquid preparations such as suspensions,adhesive preparations, film preparations, ointments, medicaldevice-containing preparations in which the active ingredient iscontained in a medical device (stent, bolt, suture thread, etc.),coating agents coated with the same, and the like). In addition, it canbe directly administered into, for example, an ischemic region of heartmuscle using blood vessel catheter or the like.

For example, when microspheres are made into injections, practicalpharmaceutical preparations for injection can be obtained by making themicrospheres into aqueous suspensions together with a dispersing agent,a preservative, a tonicity agent, a buffer agent, a pH adjusting agentand the like. Also, they are made into injections which can bepractically used as oily suspensions by dispersing together with a plantoil or its mixture with phospholipid such as lecithin, or a middle chainfatty acid triglyceride (e.g., Migliol 812, etc.).

Particle size of the microspheres, for example when used as suspensioninjections, may be within such a range that their dispersing degree andthrough-needle property are satisfied, and a range of from about 0.1 toabout 300 μm as the average particle size can be exemplified. It ispreferably within the range of from about 1 to 150 μm, more preferably aparticle size of within the range of from about 2 to 100 μm. Asdescribed above, it is preferable that the pharmaceutical composition ofthe present invention is a suspension. It is preferable that thepharmaceutical composition of the present invention is in the form offine particles. This is because the pharmaceutical composition does notgive patients too excess pain when administered through a needle whichis used for usual subcutaneous or intramuscular injection. Thepharmaceutical composition of the present invention is particularlypreferable as injections. When the microspheres are made into sterilepreparations, a method in which the entire production steps are carriedout under aseptic conditions, a method in which they are sterilized withgamma rays, a method in which an antiseptic is added and the like can beemployed, Although not particularly limited.

The action of the active ingredient of the pharmaceutical composition ofthe present invention has a sustained release property, and thesustained release period varies depending on the kind, blending amountand the like of the biodegradable polymer, it has a sustained releaseperiod of generally from 1 week to 3 months, so that it can be used as astem cell differentiation induction accelerator or an angiogenesisaccelerator by accelerating production of various endogenous repairfactors in (ischemic) organ disease regions.

Although dose of the pharmaceutical composition of the present inventionvaries depending on the kind and content of the active ingredient,dosage forms, persisting period of time of the drug release, the animalto be administered and the like, it may be an effective amount of theactive ingredient. For example, when the composition is used asmicrospheres in an ischemic region, it may be administered at a dose offrom about 0.001 mg to 500 mg, preferably from about 0.01 mg to 50 mg,per once, as the active component per adult (50 kg in body weight), fromonce a day to once in 3 months.

In addition, for the coldness, numbness, intermittent claudication, painat rest, skin ulcer or the like on the limbs in the case of ASO, Buergerdisease, diabetic neuropathy or the like, it is preferable to carry outintramuscular administration of the agent of the present invention orits persistent preparation, for example, to the diseased region or thevicinity thereof continuously for a period of approximately from once aday to 4 weeks.

For myocardial infarction, angina pectoris and the like, it ispreferable to directly carry out intramuscular administration of theagent of the present invention or its persistent preparation, forexample, to the ischemic heart muscle region or the vicinity thereof,and it is preferable to carry out the administration directly underthoractomy or using a needle-equipped blood vessel catheter or the like.As the administration period, it is preferable to carry out theintramuscular administration continuously, for example, for a period ofapproximately from once a day to 4 weeks.

In the case of osteoporosis, periodontal tissue damage, bone fracture,osteoarthritis and the like, it is preferable to administer the agent ofthe present invention or its persistent preparation alone, or by mixingit with a bone cement, a joint lubricant, a prosthetic tool or the lie,topically to the diseased region or the vicinity thereof

In the case of pulmonary hypertension, COPD and the like, it ispreferable to carry out inhalation of the agent of the present inventionor its persistent preparation as solutions for inhalation or powders forinhalation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a measured result of hollow organ formation accelerationaction of Compound 1((E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid).

FIG. 2 shows a release test result of the microsphere preparationproduced in Preparation Example 1.

FIG. 3 shows a release test result of the microsphere preparationproduced in Preparation Example 2.

FIG. 4 shows a release test result of the microsphere preparationproduced in Preparation Example 3.

FIG. 5 shows a measured result of hollow organ formation accelerationactions of Compound 3((5Z,9β,11α,13E)-17,17-propano-11,16-dihydroxy-9-chloro-20-norprost-5,13-dienoicacid) and Compound 4((11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid).

BEST MODE FOR CARRYING OUT THE INVENTION

Pharmacological tests are shown in the following as the examples of thepresent invention, but these are for thoroughly understanding thepresent invention and do not limit the scope of the present invention.In this connection, improvement of measuring accuracy and modificationof measuring sensitivity were applied as follows to the measuringmethods for evaluating the compounds of the present invention.

EXAMPLE 1

Measurement of Angiogenesis Acceleration Action (In Vitro):

Test Method:

An angiogenesis kit (manufactured by Kurabo; constituted from normalhuman umbilical cord vein vascular endothelial cells and normal humanskin fibroblasts) was cultured for 3 hours, and then the culture medium(the medium for angiogenesis use attached to the angiogenesis kit wasused as the culture medium and cultured at 37° C. under a moistenvironment of 5% carbon dioxide-95% air, and a carbon dioxide incubatorBNA-121D was used as the incubator) was changed and an agent to betested was added to each well (0.5 ml/well). The medium exchange wascarried out also on the 3rd, 6th and 8th days after commencement of theculturing and the fresh agent to be tested was added. The kind andconcentration of the agent to be tested were untreated, solvent (DMSO)0.1%, Compound 1 10⁻⁹, 10⁻⁸ and 10⁻⁷ mol/l, VEGF-A 0.1, 1 and 10 ng/mland HGF 0.1, 1 and 10 ng/ml, and the culturing was carried out by using3 wells for each concentration. Fixation was carried out 10 days afterthe commencement of culturing, and staining of hollow organ withanti-CD31 antibody was carried out by using a hollow organ staining kit(manufactured by Kurabo). As the evaluation, Chalkley Grid (a grid lens,manufactured by Kurabo) was mounted on the eyepiece of a microscope, andthe hollow organ formation was evaluated by counting intersections ofthe randomly arrange points of Chalkley Grid with the formed holloworgan (by referring to the evaluation method of J. Pathol., 177, 275-283(1995)). The counting was carried out at 12 positions per well, and thetotal was calculated.

As the agent to be tested, Compound 1((E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid) was dissolved in dimethyl sulfoxide (DMSO) to prepare 10⁻⁴, 10⁻⁵and 10⁻⁶ mol/l solutions which were then used by diluting 1/1000 timewith the culture medium.

As the VEGF-A (vascular endothelial growth factor-A), the 2 μg/mlsolution of VEGF contained in the angiogenesis control reagent kit ofKurabo was diluted with the culture medium and used.

As the HGF (human hepatocyte growth factor), the 5 μg/ml solution of HGFpurchased from R & D System-Funakoshi was diluted with the culturemedium and used.

Statistical Analysis Method:

The counts of hollow organ formation in the untreated wells werecompared with those of the wells treated with respective concentrationsof test samples by Dunnett's test (two-sided test). The significancelevel was set to 5%.

In this connection, the data were shown by average value of 3 wells andstandard deviation.

The test results are shown in FIG. 1.

Results:

Compound 1 accelerated hollow organ formation statisticallysignificantly by 10⁻⁸ and 10⁻⁷ mol/l. In addition, the VEGF-A and HGFaccelerated hollow organ formation at a concentration of 10 ng/ml. Basedon the above results, it was revealed that Compound 1 has anangiogenesis accelerating effect having a strength equivalent to thepositive control agents VEGF-A and HGF in a co-culture system of humanvascular endothelial cells and human fibroblasts.

EXAMPLE 2

Measurement of Endogenous Repair Factor (HGF, VEGF) Protein ProducingAction (In Vitro):

Test Method:

An angiogenesis kit (manufactured by Kurabo; constituted from normalhuman umbilical cord vein vascular endothelial cells and normal humanskin fibroblasts) was cultured for 3 hours, and then the culture medium(the medium for angiogenesis use attached to the angiogenesis kit wasused as the culture medium and cultured at 37° C. under a moistenvironment of 5% carbon dioxide-95% air, and a carbon dioxide incubatorBNA-121D was used as the incubator) was changed and an agent to betested was added to each well (0.5 ml/well). The agent to be tested weresolvent (DMSO) 0.1% and Compound 1 10⁻⁷ mol/l, and the culturing wascarried out by using 3 wells for each. Culture supernatants werecollected before commencement of the culturing and 1, 2, 6, 24, 48 and72 hours after the commencement. HGF and VEGF protein concentrations inthe culture supernatants were measured by using an ELISA kit (R % Dsystem-Funakoshi).

As the agent to be tested, Compound 1((E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid) was dissolved in dimethyl sulfoxide (DMSO) to prepare 10⁻⁴ mol/lsolution which was then used by diluting 1/1000 time with the culturemedium.

Statistical Analysis Method:

Results of the solvent control wells were compared with those of thewells treated with the agent to be tested by Dunnett's test (two-sidedtest). The significance level was set to 5%.

In this connection, the data were shown by average value of 3 wells andstandard deviation.

The test results after 72 hours of the culturing are shown in Table 1.TABLE 1 Agent tested HGF (pg/ml) VEGF (pg/ml) Solvent control 110 ± 162004 ± 76 (DMSO) Compound 1 518 ± 15*** 3034 ± 30******p < 0.00 1 vs. solvent controlResults:

Compound 1 accelerated production of HGF protein and VEGF proteinstatistically significantly in comparison with the solvent control, by72 hours of culturing at a concentration of 10⁻⁷ mol/l.

EXAMPLE 3

Production of Persistent Preparations:

Preparation Example 1

A dichloromethane (1 ml) solution of 100 mg of a polylacticacid-glycolic acid copolymer (hereinafter referred to as “PLGA”)(polylactic acid:glycolic acid=1:1 (mol %), weight average molecularweight 40,000, PLGA5-1, manufactured by Mitsui Kagaku) and Compound 1 (5mg) was prepared. An 0/W emulsion was prepared by adding the solutionprepared in the above to 300 ml of 0.1% polyvinyl alcohol (NacalaiTesque) aqueous solution (pH 3.0, adjusted with 1 N hydrochloric acid)which was stirred at 5,000 rpm using TK Robomix (Tokushu Kiki, MARK II2.5 type), and stirring the mixture at room temperature for 3 minutes.This O/W emulsion was stirred at room temperature for 2 hours toevaporate dichioromethane, and the oil phase was solidified and thencentrifuged at 3,000 rpm for 10 minutes using a centrifuge (Hitachi,O5PR-22). The supernatant was discarded, and the residue was dispersedin distilled water for injection (35 ml) and then centrifuged at 3,000rpm for 10 minutes using the centrifuge. The supernatant was discarded,and the residue was dispersed in 0.2% Tween 80 solution (35 ml) and thencentrifuiged at 3,000 rpm for 10 minutes using the centrifuige. Thesupernatant was discarded, and the residue was dispersed in distilledwater for injection (35 ml) and then again centrifuiged at 3,000 rpm for10 minutes using the centrifuge. Finally discarding the supernatant, theprecipitate was soaked in dry ice-methanol, frozen and then dried undera reduced pressure, thereby producing a microsphere preparation ofCompound 1.

Preparation Example 2

A dichloromethane (1 ml) solution of 100 mg of a polylacticacid-glycolic acid copolymer (hereinafter referred to as “PLGA”)(polylactic acid:glycolic acid=1:1 (mol %), weight average molecularweight 20,000, PLGA5020, Wako Pure Chemical Industries) and Compound 1(5 mg) was prepared. Thereafter, the same operation of PreparationExample 1 was carried out to produce a microsphere preparation ofCompound 1.

Preparation Example 3

A dichloromethane (3 ml) solution of 100 mg of a polylacticacid-glycolic acid copolymer (to be referred to as PLGA hereinafter)(polylactic acid:glycolic acid=1:1 (mol %), weight average molecularweight 40,000, PLGA5-1, manufactured by Mitsui Kagaku) and Compound 1 (5mg) was prepared. Thereafter, the same operation of Preparation Example1 was carried out to produce a microsphere preparation of the Compound1.

Preparation Test Example 1

Measurement of Inclusion Efficiency:

The microspheres produced in Preparation Examples 1, 2 and 3(respectively about 10 mg) were mixed with an acetonitrile solutioncontaining an appropriate internal standard and dissolved by carryingout an ultrasonic treatment. Compound 1 content of each of the solutionswas measured by a high performance liquid chromatography, and inclusionefficiency of Compound 1 in the microsphere was calculated by thefollowing formula.Inclusion efficiency (%)=(content found/content calculated)×100

As a result, the microsphere preparation of Preparation Example 1 showedan inclusion efficiency of 70.9%, the microsphere preparation ofPreparation Example 2 showed an inclusion efficiency of 100% and themicrosphere preparation of Preparation Example 3 showed an inclusionefficiency of 74.3%.

Preparation Test Example 2: In Vitro Release Test:

Each of the microsphere preparations produced in Preparation Examples 1,2 and 3 was added to 0.2% Tween 80 1/15 M phosphate buffer pH 6.8 to aconcentration of 100 μg/ml as the agent and uniformly dispersed byultrasonic treatment using Vortex. This was dispensed at 1 ml intocontainers and put into a 37° C. constant temperature oven. Thecontainers were periodically sampled out and centrifuged at 12,000 rpmfor 5 minutes, and the residual amount of Compound 1 in microspheres ofthe pellet was measured by a high performance liquid chromatography(HPLC).

Results of the microsphere preparation produced in Preparation Example 1are shown in FIG. 2, and results of the microsphere preparation producedin Preparation Example 2 in FIG. 3, and results of the microspherepreparation produced in Preparation Example 3 in FIG. 4.

In this connection, the residual ratio in FIGS. 2, 3 and 4 means a ratioof Compound 1 remaining in microspheres to the initial.

As a result, the microsphere preparation of Preparation Example 1released about 40% of the agent during 14 days, and the microspherepreparation of Preparation Example 2 released the whole amount duringabout 10 days. The microsphere preparation of Preparation Example 3released about 60% during 28 days.

EXAMPLE 4

Angiogenesis Test (In Vivo Test) Using Rat Leg Ischemia(Arteriosclerosis Obliterans (ASO)) Model:

Leg ischemia model was prepared by ligating rat left femoral artery.Blood flow in hind legs was measured by using Laser Doppler Imager (MoorInstruments) 2 weeks after the preparation, and the animals were dividedinto 6 groups (n=5) in such a manner that the average value of bloodflow became almost uniform.

Starting on the next day of the grouping, 0.1 ml/site, 2 sites, of theagent to be tested was administered by intramuscular injection into theleft femoral adductor, for once a week, 4 times in total as a solutionto be tested. One week after completion of the final administration,blood flows of hind legs were measured by using Laser Doppler Imager(Moor Instruments), and blood flows of the treated leg (left leg) anduntreated leg (right leg) were compared and examined. The results oftreated leg/untreated leg (%) are shown in Table 2.

In this connection, construction of the solution to be tested is asfollows. Solvent (control) group: 0.2 w/v/o Tween 80 solution (0.2 ml).

Polymer Group:

The polylactic acid-glycolic acid copolymer used in Preparation Example2 was suspended in 0.2 w/v % Tween 80 solution (0.2 ml). In thisconnection, amount of the polylactic acid-glycolic acid copolymer isidentical to the amount contained in the Compound 1 MS (1 mg).

Compound 1 (1 mg) Group:

Compound 1 (1 mg) was suspended in 0.2 w/v % Tween 80 solution (0.2 ml).

Compound 1 MS (0.01 mg) Group:

The microsphere preparation produced in Preparation Example 2 containing0.01 mg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.2ml).

Compound 1 MS (0.1 mg) Group:

The microsphere preparation produced in Preparation Example 2 containing0.1 mg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.2ml).

Compound 1 MS (1 mg) Group:

The microsphere preparation produced in Preparation Example 2 containing1 mg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.2ml). TABLE 2 Treated leg/untreated leg Solution to be tested blood flowratio (%) Control 67.3 ± 5.1 Polymer 68.7 ± 4.1 Compound 1 (1 mg) 76.2 ±2.8**^(##) Compound 1 MS (0.01 mg) 74.0 ± 7.1 Compound 1 MS (0.1 mg)78.3 ± 4.6**^(##) Compound 1 MS (1 mg) 89.0 ± 4.4**^(##$$)**significant difference from Control at p<0.01 (Student's t-test)^(##)significant difference from Polymer at p<0.01 (Student's t-test)^($$)significant difference from Compound 1 (1 mg) at p<0.01 (Student'st-test)Results:

Although significant recovery of blood flow against Control was foundeven by the administration of Compound 1 (I mg) alone, further strongerblood flow improving effect than Compound 1 (1 mg) was observed by theadministration of the microsphere (MS) preparation (Preparation Example2) of Compound 1.

In comparison with the polymer group, dose-correlative blood flowimproving effect was found in Compound 1 MS preparation, and significantblood flow improving effect action was found in Compound 1 MS (0.1 mg)and Compound 1 MS (1 mg).

EXAMPLE 5

Angiogenesis Test Using Rat Leg Ischemia (Arteriosclerosis Obliterans(ASO)) Model; Determination of Minimum Effective Dose (In Vivo Test):

Leg ischemia model was prepared by ligating rat left femoral artery.Blood flow in hind legs was measured by using Laser Doppler Imager (MoorInstruments) 1 week after the preparation, and the animals were dividedinto 4 groups (n=5) in such a manner that the average value of bloodflow became almost uniform.

Starting on the next day of the grouping, 0.1 mi/site, 2 sites, of theagent to be tested was administered by intramuscular injection into theleft femoral adductor (polymer group, Compound 1 MS group), for once aweek, 4 times in total as a solution to be tested. One week aftercompletion of the final administration, blood flows of hind legs weremeasured by using Laser Doppler Imager (Moor Instruments), and bloodflows of the treated leg (left leg) and untreated leg (right leg) werecompared and examined. The results of treated leg/untreated leg (%) areshown in Table 3.

In this connection, construction of the solution to be tested is asfollows.

Polymer Group:

The polylactic acid-glycolic acid copolymer used in Preparation Example2 was suspended in 0.2 w/v % Tween 80 solution (0.2 ml). In thisconnection, amount of the polylactic acid-glycolic acid copolymer isidentical to the amount contained in Compound 1 MS (0.1 mg).

Compound 1 MS (0.03 mg) Group:

The microsphere preparation produced in Preparation Example 2 containing0.03 mg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.2ml).

Compound 1 MS (0.1 mg) Group:

The microsphere preparation produced in Preparation Example 2 containing0.1 mg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.2ml).

Compound 1 MS (0.3 mg) Group:

The microsphere preparation produced in Preparation Example 2 containing0.3 mg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.2ml). TABLE 3 Treated leg/untreated leg Solution to be tested blood flowratio (%) Polymer 57.3 ± 4.5 Compound 1 MS (0.03 mg) 65.1 ± 3.7*Compound 1 MS (0.1 mg) 68.4 ± 3.2** Compound 1 MS (0.3 mg) 72.7 ± 4.6***p<0.05 vs. Polymer (Student's t-test)**p<0.01 vs. Polymer (Student's t-test)Results:

In comparison with the polymer group, dose-correlative blood flowimproving effect was found in the microsphere (MS) preparation ofCompound 1, and significant blood flow improving effect action was foundin Compound 1 MS (0.03 mg). This, since significant blood flow improvingeffect action was not found in Compound 1 MS (0.01 mg) in Example 4, itwas suggested that the minimum effective dose is 0.03 mg.

EXAMPLE 6

Angiogenesis Test Using Rat Leg Ischemia (Arteriosclerosis Obliterans(ASO)) Model; Usefulness of Topical Administration (In Vivo Test):

Leg ischemia model was prepared by ligating rat left femoral artery.Blood flow in hind legs was measured by using Laser Doppler Imager (MoorInstruments) 1 week after the preparation, and the animals were dividedinto 4 groups (n=5) in such a manner that the average value of bloodflow became almost uniform.

Starting on the next day of the grouping, 0.1 ml/site, 2 sites, of theagent to be tested was administered by intramuscular injection intoischemic regions of the left femoral adductor (polymer group, Compound 1group, Compound 1 MS group) and normal region of the right shoulderupper arm (Compound 1 MS group), for once a week, 4 times in total as asolution to be tested. One week after completion of the finaladministration, blood flows of hind legs were measured by using LaserDoppler Imager (Moor Instruments), and blood flows of the treated leg(left leg) and untreated leg (right leg) were compared and examined. Theresults of treated leg/untreated leg (%) are shown in Table 4.

In this connection, construction of the solution to be tested is asfollows.

Polymer Group:

The polylactic acid-glycolic acid copolymer used in Preparation Example2 was suspended in 0.2 w/v % Tween 80 solution (0.2 ml). In thisconnection, amount of the polylactic acid-glycolic acid copolymer isidentical to the amount contained in Compound 1 MS (1 mg).

Compound 1 (0.1 mg) Group:

Compound 1 (0.1 mg) was suspended in 0.2 w/v % Tween 80 solution (0.2ml).

Compound 1 MS (0.1 mg) Group:

The microsphere preparation produced in Preparation Example 2 containing0.1 mg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.2ml). TABLE 4 Treated leg/untreated leg Solution to be tested blood flowratio (%) Polymer^(a)) 57.3 ± 4.5 Compound 1 (0.1 mg)^(a)) 60.5 ± 3.1Compound 1 MS (0.1 mg)^(b)) 56.9 ± 5.6 Compound 1 MS (0.1 mg)^(a))  68.4± 3.2*^(a))Intramuscular administration into the ischemic left femoral part^(b))Intramuscular administration into right normal upper arm**p <0.01 vs. Polymer (Student's t-test)Results:

Although significant increase in the blood flow was not found by theintramuscular administration of Compound 1 MS (0.1 mg) into the rightnormal upper arm, significant increase in the blood flow was found bythe intramuscular administration of the same dose into the ischemic leftfemoral part. Based on this, it was suggested that efficacy of Compound1 is not mediated by blood flow, but its topical administration intoischemic regions is important. In addition, since significant blood flowincreasing action was not observed by the topical administration ofCompound 1 into ischemic region, efficacy of the persistent preparation(MS) was suggested.

EXAMPLE 7

Angiogenesis Test Using Rat Leg Ischemia (Arteriosclerosis Obliterans(ASO)) Model; Vasodilation Action and Angiogenesis Acceleration Action(In Vivo Test):

Leg ischemia model was prepared by ligating rat left femoral artery.Blood flow in hind legs was measured by using Laser Doppler Imager (MoorInstruments) 1 week after the preparation, and the animals were dividedinto 2 groups (n=5) in such a manner that the average value of bloodflow became almost uniform.

Starting on the next day of the grouping, the solution to be tested wasadministered by intramuscular injection into the left femoral adductor(polymer group, Compound 1 MS group) for once a week, 4 times in total.Blood flows of hind legs were measured 3 days after the secondadministration (on the 10th day after grouping) and after 1 week and 2weeks from the completion of the final administration, by using LaserDoppler Imager (Moor Instruments), and blood flows of the treated leg(left leg) and untreated leg (right leg) were compared and examined. Theresults of treated leg/untreated leg (%) are shown in Table 5.

In this connection, construction of the solution to be tested is asfollows.

Polymer Group:

The polylactic acid-glycolic acid copolymer used in Preparation Example2 was suspended in 0.2 w/v % Tween 80 solution (0.2 ml). In thisconnection, amount of the polylactic acid-glycolic acid copolymer isidentical to the amount contained in Compound 1 MS (0.3 mg).

Compound 1 MS (0.3 mg) Group:

The microsphere preparation produced in Preparation Example 2 containing0.3 mg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.2ml). TABLE 5 Treated leg/untreated leg blood flow ratio (%) After 3 daysAfter 1 week Solution Before 1st of 2nd of 4th After 2 weeks to beadministra- administra- administra- 4th of tested tion tion tionadministration Polymer 36.7 ± 4.2 49.8 ± 5.8 57.3 ± 4.5 61.3 ± 2.5Compound 36.6 ± 4.3 59.9 ± 3.7* 72.7 ± 4.6* 72.4 ± 5.7** 1 MS (0.3 mg)*p<0.05 vs. Polymer (Student's t-test)**p<0.01 vs. Polymer (Student's t-test)Results:

In comparison with the polymer-administered group, a significant bloodflow increase of about 10% was observed at the ischemic region byCompound 1 MS (0.3 mg) even on the 10th day after the 1st administration(3 days after the 2nd administration). Since a period of about 4 weeksis necessary for angiogenesis and Compound 1 is under release at theischemic topical region 3 days after the 2nd administration, it wassuggested that this improving effect is a blood flow increasing effectby direct actions such as vasodilation action and platelet agglutinationinhibitory action of slow-released Compound 1. In addition, asignificant blood flow increase action of about 11% was also observedafter 1 week and 2 weeks of the final administration. It was consideredbased on this that release of Compound 1 from the microspherepreparation was completely disappeared after one week of the finaladministration, and it was suggested that this effect is not the directactions of Compound 1 (vasodilation action, platelet agglutinationinhibitory action, etc.), but an effect by the angiogenesis action.

EXAMPLE 8

Angiogenesis Test (In Vivo Test) Using Mouse Sponge TransplantationModel:

Under anesthesia, dorsal part of a mouse was incised and a discusurethane sponge (about 5 mm in thickness and 13 mm in diameter) wasembedded therein. Administration of the agent was carried out bytopically administering it directly into the sponge once a day for atotal of 14 times starting on the day of the sponge transplantationmodel preparation, or on the operation-completed day and 7th daythereafter. On the 15th day after the sponge transplantation, the spongecontaining granulation tissue was extracted and observed with the nakedeye, and then its wet mass was measured. In addition, this was mixedwith distilled water of 4 times larger amount than the wet mass,homogenized and centrifuged, and then the supernatant was subjected tothe measurement of the hemoglobin content using Hemoglobin β-Test Wako(manufactured by Wako Pure Chemical Industries). Results of measurementof the total hemoglobin content in the extracted sponges are shown inTable 6.

In this connection, construction of the solution to be tested is asfollows.

Polymer Group:

The polylactic acid-glycolic acid copolymer used in Preparation Example2 was suspended in 0.2 w/v % Tween 80 solution (0.05 ml). In thisconnection, amount of the polylactic acid-glycolic acid copolymer isidentical to the amount contained in Compound 1 MS (200 μg).

Compound 1 (20 μg) Group:

Compound 1 (20 μg) was suspended in 0.2 w/v % Tween 80 solution (0.05ml).

Compound 1 (40 μg) Group:

Compound 1 (40 μg) was suspended in 0.2 w/v % Tween 80 solution (0.05ml)

Compound 1 (200 μg) Group:

Compound 1 (200 μg) was suspended in 0.2 w/v % Tween 80 solution (0.05ml).

Compound 1 MS (200 μg) Group:

The microsphere preparation produced in Preparation Example 2 containing200 μg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.05ml).

Compound 1 MS (400 μg) Group:

The microsphere preparation produced in Preparation Example 2 containing400 μg of Compound 1 was suspended in 0.2 w/v % Tween 80 solution (0.05ml).

Compound 3 (20 μg) Group:

Compound 3 (20 μg) was suspended in 0.2 w/v % Tween 80 solution (0.05ml). TABLE 6 Solution to Sponge wet Hemoglobin content be tested weight(g) (mg/g wet tissue) Polymer^(b)) 0.4542 ± 0.0303 1.973 ± 0.564Compound 1 (20 μg)^(a)) 0.3843 ± 0.0681 2.403 ± 0.533 Compound 1 (40μg)^(a)) 0.5136 ± 0.0938 3.010 ± 0.808 Compound 1 (200 μg)^(b)) 0.4123 ±0.0320 1.964 ± 0.289 Compound 1 MS (200 μg)^(b)) 0.5317 ± 0.1413 3.523 ±0.482** Compound 1 MS (400 μg)^(b)) 0.5655 ± 0.1130 4.822 ± 1.218**Compound 3 (20 μg)^(a)) 0.5193 ± 0.0792 4.588 ± 0.488**^(a))once a day, 14 days of repeated administration^(b))two administrations at 7 day intervals^(**)p<0.01 vs. Polymer (Dunnett's test)Results:

Formation of granulation was found in the sponges administered withCompound 1 MS (200 μg) (two administrations at 7 day intervals) andCompound 1 MS(400 μg) (two administrations at 7 day intervals), and theyturned pale red, red or dark brown. In addition, when hemoglobinconcentration in the granulation tissue formed in each sponge wasmeasured, significant increase in the concentration of hemoglobin incomparison with the polymer group was observed so that the angiogenesiseffect was confirmed. On the other hand, in the case of Compound 1 (20μg) (14 days of repeated administration) and Compound 1 (40 μg) (14 daysof repeated administration), hemoglobin concentration in the granulationtissue formed in each sponge showed an increasing tendency, but is not asignificant increase. Also, hemoglobin concentration in the granulationtissue formed in the sponge did not increase in the case of Compound 1(200 μg) (two administrations at 7 day intervals). Based on this, it wasfound that the slow release preparation of Compound 1 (Compound 1 MS) isparticularly useful in inducing angiogenesis. Also, formation ofgranulation was found in the sponge administered with Compound 3(20 μg)(14 days of repeated administration), and they turned pale red, red ordark brown. In addition, when hemoglobin concentration in thegranulation tissue formed in the sponge was measured, significantincrease in the concentration of hemoglobin in comparison with thepolymer group was observed so that the angiogenesis effect wasconfirmed.

EXAMPLE 9

Measurement of Angiogenesis Acceleration Action (In Vitro):

Test Method:

An angiogenesis kit (manufactured by Kurabo;. constituted from normalhuman umbilical cord vein vascular endothelial cells and normal humanskin fibroblasts) was cultured for 3 hours, and then the culture medium(the medium for angiogenesis use attached to the angiogenesis kit wasused as the culture medium and cultured at 37° C. under a moistenvironment of 5% carbon dioxide-95% air, and a carbon dioxide incubatorBNA-121D was used as the incubator) was changed and an agent to betested was added to each well (0.5 ml/well). The medium exchange wascarried out also on the 3rd, 6th and 8th days after commencement of theculturing and the fresh agent to be tested was added. The kind andconcentration of the agent to be tested were untreated; DMSO: 0.1%;α-CD: 0.0118, 0.118 and 1.18 mg/ml; PGE2-αCD: 1, 10 and 100 nmol/l;Compound 3 (EP2 agonist), Compound 4 (EP4 agonist), EP1 agonist and EP3agonist: 1, 10 and 100 nmol/l, VEGF: 0.1, 1 and 10 ng/ml; and HGF: 0.1,1 and 10 ng/ml, and culturing was carried out by using 3 wells for eachconcentration. Fixation was carried out 10 days after the commencementof culturing, and staining of hollow organ with anti-CD31 antibody wascarried out by using a hollow organ staining kit (manufactured byKurabo). As the evaluation, Chalkley Grid (a grid lens, Kurabo) wasmounted on the eyepiece of a microscope, and the hollow organ formationwas evaluated by counting intersections of the randomly arrange pointsof Chalkley Grid with the formed hollow organ (by referring to theevaluation method of J. Pathol., 177, 275-283 (1995)). The counting wascarried out at 12 positions per well, and the total was calculated.

In this connection, Compound 3 as an EP2 agonist((5Z,9β,11α,13E)-17,17-propano-11;16-dihydroxy-9-chloro-20-norprost-5,13-dienoicacid) was dissolved in dimethyl sulfoxide (DMSO) to prepare 10⁻⁴, 10⁻⁵and 10⁻⁶ mol/l solutions which were then used by diluting 1/1000 timewith the culture medium.

Compound 4 as an EP4 agonist((11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid) was dissolved in dimethyl sulfoxide (DMSO) to prepare 10⁻⁴, 10⁻⁵and 10⁻⁶ mol/l solutions which were then used by diluting 1/1000 timewith the culture medium.

The EP1 agonist((13E)-(11α,15S,17S)-2,5-ethano-6,9-dioxo-11,15-dihydroxy-17,20-dimethylprost-13-enoicacid; the compound described in Example 1 in the specification ofJP-A-11-322709) was dissolved in dimethyl sulfoxide (DMSO) to prepare10⁻⁴, 10⁻⁵ and 10⁻⁶ mol/l solutions which were then used by diluting1/1000 time with the culture medium.

The EP3 agonist (11α, 15α-dimethoxy-9-oxoprost-5Z, 13E-dienoic acid; thecompound described in Example 1 in the specification of WO98/34916) wasdissolved in dimethyl sulfoxide (DMSO) to prepare 10⁻⁴, 10⁻⁵ and 10⁻⁶mol/l solutions which were then used by diluting 1/1000 time with theculture medium.

The PGE2-αCD was dissolved in distilled water for injection to prepare10⁻⁴, 10⁻⁵ and 10⁻⁶ mol/l solutions which were then used by diluting1/1000 time with the culture medium.

As the VEGF (vascular endothelial growth factor), the 2 μg/ml solutionof VEGF contained in the angiogenesis control reagent kit of Kurabo wasdiluted with the culture medium and used.

As the HGF (human hepatocyte growth factor), the 5 1g/ml solution of HGFpurchased from R & D System-Funakoshi was diluted with the culturemedium and used.

Statistical Analysis Method:

The counts of hollow organ formation in the untreated wells werecompared with those of the wells treated with respective concentrationsof test samples by Dunnett's test (two-sided test). The significancelevel was set to 5%.

In this connection, the data were shown by average value of 3 wells andstandard deviation.

The test results are shown in FIG. 5.

Results:

The PGE2-αCD, Compound 3 (EP2 agonist) and Compound 4 (EP4 agonist)significantly accelerated hollow organ formation at concentrations of 10nmol/l and 100 nmol/l. In addition, the VEGF and HGF accelerated holloworgan formation at a concentration of 10 ng/ml. However, the EP1agonist, EP3 agonist and α-CD did not exert influence upon hollow organformation.

EXAMPLE 10

Measurement of Endogenous Repair Factor Releasing Action (In Vitro):

Test Method:

An angiogenesis kit (manufactured by Kurabo; constituted from normalhuman umbilical cord vein vascular endothelial cells and normal humanskin fibroblasts) was cultured for 3 hours, and then the culture medium(the medium for angiogenesis use attached to the angiogenesis kit wasused as the culture medium and cultured at 37° C. under a moistenvironment of 5% carbon dioxide-95% air, and a carbon dioxide incubatorBNA-121D was used as the incubator) was changed and an agent to betested was added to each well (0.5 ml/well). HGF and VEGF concentrationsin the culture supernatants were measured 3 days after the commencementof the culturing. Kinds and concentrations of the agent to be testedwere untreated; DMSO: 0.1%; PGE1-αCD: 100 nmol/; PGE2-αCD: 100 nmol/l;and Compound 3 (EP2 agonist) and Compound 4 (EP4 agonist): 100 nmol/l,and the culturing was carried out by using 3 wells for eachconcentration. The HGF and VEGF concentrations were measured by using anELISA kit (R % D system-Funakoshi).

In this connection, Compound 3 as an EP2 agonist((5Z,9β,11α,13E)-17,17-propano-11,16-dihydroxy-9-chloro-20-norprost-5,13-dienoicacid) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10⁻⁴ mol/lsolution which was then used by diluting 1/1000 time with the culturemedium.

Compound 4 as an EP4 agonist((11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10⁻⁴ mol/lsolution which was then used by diluting 1/1000 time with the culturemedium.

The PGE1-αCD and PGE2-αCD were dissolved in distilled water forinjection to prepare 10⁻⁴ mol/l solutions which were then used bydiluting 1/1000 time with the culture medium.

Statistical Analysis Method:

HGF and VEGF concentrations in the supernatants of the solvent controlgroup were compared with those of the agent-treated groups by Dunnett'stest (two-sided test). The significance level was set to 5%.

In this connection, the data were shown by average value of 3 wells andstandard deviation.

The test results after 72 hours of the culturing are shown in Table 7.TABLE 7 Agent tested HGF (pg/ml) VEGF (pg/ml) Untreated  110 ± 16 2004 ±76 PGE1-αCD 2043 ± 77*** 2710 ± 50*** PGE2-αCD 1735 ± 95*** 3032 ±165**** EP2 agonist (Compound 3) 1865 ± 48*** 2762 ± 51*** EP4 agonist(Compound 4)  379 ± 12*** 2936 ± 69******p<0.001 vs. untreated (Dunnett's test)Results:

The PGE1-αCD, PGE2-αCD, Compound 3 (EP2 agonist) and Compound 4 (EP4agonist) significantly increased HGF and VEGF concentrations insupernatants at a concentration of 100 nmol/l.

1.-16. (canceled)
 17. A method for accelerating production of anendogenous repair factor in a mammal, which comprises administering to amammal an effective amount of one or at least two selected from a PGI2agonist, an EP2 agonist and an EP4 agonist.
 18. A method for preventingand/or treating organ diseases in a mammal, which comprisesadministering to a mammal an effective amount of one or at least twoselected from a PGI2 agonist, an EP2 agonist and an EP4 agonist. 19.(canceled)
 20. (canceled)
 21. A pharmaceutical composition whichcomprises the endogenous repair factor production accelerator comprisingone or least two selected from a PGI2 agonist, an EP2 agonist and an EP4agonist in combination with one or at least two selected from ananti-thrombus agent, a circulation improving agent, a bronchial smoothmuscle dilator, an anti-inflammatory drug, a local anesthetic, ananalgesic, a bone cement, an joint lubricant, a PG derivative, anendogenous repair factor protein, an endogenous repair factor gene and astem cell.
 22. The method according to claim 17, wherein the endogenousrepair factor is a vascular endothelial growth factor, a hepatocytegrowth factor, a fibroblast growth factor, a transformation growthfactor-β, a platelet derived growth factor, a bone morphogenetic proteinor an epidermal growth factor.
 23. The method according to claim 17,wherein stem cell differentiation is induced.
 24. The method accordingto claim 17, wherein angiogenesis is accelerated.
 25. The methodaccording to claim 17, wherein the PGI2 agonist, the EP2 agonist or theEP4 agonist is administered as a persistent prerparation which furthercomprises a biodegradable polymer.
 26. The method according to claim 25,wherein the persistent preparation is a microsphere preparation, amicrocapsule preparation or a nanosphere preparation.
 27. The methodaccording to claim 17, wherein organ diseases are prevented and/ortreated.
 28. The method according to claim 27, wherein the organ diseaseis an ischemic organ disease, a liver disease, a kidney disease, a lungdisease, a pancreas disease, a bone disease, a digestive organ disease,a nerve degeneration disease, a diabetic complication, a vascularendothelial cell disease, a heart disease, a dental disease, decubitus,glaucoma or alopecia.
 29. The method according to claim 28, wherein theischemic organ disease is arteriosclerosis obliterans, Buerger disease,Raynaud disease, myocardial infarction, angina pectoris, diabeticneuropathy, spinal canal stenosis, cerebrovascular accidents, cerebralinfarction, pulmonary hypertension, bone fracture or Alzheimer disease.30. The method according to claim 17, wherein the PGI2 agonist is acompound represented by formula (I):

is

wherein R¹ represents hydrogen or C1-4 alkyl; R² represents (i)hydrogen, (ii) C1-8 alkyl, (iii) phenyl or C4-7 cycloalkyl, (iv) a 4- to7-membered monocyclic ring containing one nitrogen atom, (v) C1-4 alkylsubstituted with a benzene ring or C4-7 cycloalkyl, or (vi) C1-4 alkylsubstituted with a 4- to 7-membered monocyclic ring containing onenitrogen atom; R³ represents (i) C1-8 alkyl, (ii) phenyl or C4-7cycloalkyl, (iii) a 4- to 7-membered monocyclic ring containing onenitrogen atom, (iv) C1-4 alkyl substituted with a benzene ring or C4-7cycloalkyl, or (v) C1-4 alkyl substituted with a 4- to 7-memberedmonocyclic ring containing one nitrogen atom; e represents an integer offrom 3 to 5; f represents an integer of from 1 to 3; p represents aninteger of from 1 to 4; r represents an integer of from 1 to 3; qrepresents an integer of 1 or 2, and wherein, when

is the group represented by (iii) or (iv), -(CH₂)_(p)- andαCH—(CH₂)_(s)- are bound to the position of a or b on the ring, and therings in R² and R³ may be substituted with 1 to 3 of C1-4 alkyl, C1-4alkoxy, halogen, nitro or trihalomethyl, or a salt thereof.
 31. Themethod according to claim 30, wherein the PGI2 agonist is (1)(E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid, or (2)(Z)-[5-[2-[1-phenyl-1-(3-pyridyl)methylideneaminoxy]ethyl]-7,8-dihydronaphthalen-1-yloxy]aceticacid.
 32. The method according to claim 17, wherein the PGI2 agonist is(1)(±)-(1R,2R,3aS,8bS)-2,3,3a,8b-terahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butanoicacid sodium salt, (2)5-{(3aR,4R,6aS)-5-hydroxy-4-[(1E,3S)-3-hydroxy-3-(cis-4-propylcyclohexyl)prop-1-enyl-3,3a,4,5,6,6a-hexahydrocyclopenta[b]pyrrol-2-yl}pentanoicacid methyl ester, or (3) (5E)-5-[(3aS,4R,5R,6aS)-4-[(1E,3S)-3-cyclopentyl-3-hydroxyprop-1-enyl]-5-hydroxyhexahydropentalene-2(1H)-ylidene]pentanoicacid.
 33. The method according to claim 17, wherein the EP2 agonist is acompound represented by formula (I-a):

wherein R^(a) represents carboxyl or hydroxymethyl; R^(1a) representsoxo, methylene or halogen; R^(2a) represents hydrogen, hydroxyl or C1-4alkoxy; R^(3a) represents hydrogen, C1-8 alkyl, C2-8 alkenyl, C2-8alkynyl, or C1-8 alkyl, C2-8 alkenyl or C2-8 alkynyl substituted with 1to 3 of the following groups (1) to (5): (1) halogen, (2) C1-4 alkoxy,(3) C3-7 cycloalkyl, (4) phenyl, (5) phenyl substituted with 1 to 3halogen, C1-4 alkyl, C1-4 alkoxy, nitro or trifluoromethyl; narepresents 0 or an integer of from 1 to 4;

represents a single bond or a double bond;

represents a double bond or a triple bond; and

represents a single bond, a double bond or a triple bond, and wherein(1) when the 5-6 position represents a triple bond, the 13-14 positiondoes not represent a triple bond, and (2) when the 13-14 positionrepresents a double bond, the a double bond represents E form, Z form orEZ form, a salt thereof, a pro drug thereof or a cyclodextrin clathratethereof.
 34. The method according to claim 33, wherein the EP2 agonistis(5Z,9β,11α,13E)-17,17-propano-11,16-dihydroxy-9-chloro-20-norprost-5,13-dienoicacid.
 35. The method according to claim 17, wherein the EP4 agonist is acompound represented by formula (I-b):

wherein R^(1b) represents hydroxyl, C1-6 alkoxy or —NR^(6b)R^(7b);R^(6b) and R^(7b) each independently represents hydrogen or C1-4 alkyl;R^(2b) represents oxo, halogen or —O—COR^(8b); R^(8b) represents C1-4alkyl, phenyl or phenyl(C1-4 alkyl); R^(3b) represents hydrogen orhydroxyl; R^(4ab) and R^(4bb) each independently represents hydrogen orC1-4 alkyl; R^(5b) represents phenyl substituted with a group of thefollowing i) to iv): i) 1 to 3 of C1-4 alkoxy-C1-4 alkyl, C2-4alkenyloxy-C1-4 alkyl, C2-4 alkynyloxy-C1-4 alkyl, C3-7cycloalkyloxy-C1-4 alkyl, C3-7 cycloalkyl(C1-4 alkoxy)-C1-4 alkyl,phenyloxy-C1-4 alkyl, phenyl-C1-4 alkoxy-C1-4 alkyl, C1-4 alkylthio-C1-4alkyl, C2-4 alkenylthio-C1-4 alkyl, C2-4 alkynylthio-C1-4 alkyl, C3-7cycloalkylthio-C1-4 alkyl, C3-7 cycloalkyl(C1-4 alkylthio)-C1-4 alkyl,phenylthio-C1-4 alkyl, or phenyl-C1-4 alkylthio-C1-4 alkyl, ii) C1-4alkoxy-C1-4 alkyl and C1-4 alkyl, C1-4 alkoxy-C1-4 alkyl and C1-4alkoxy, C1-4 alkoxy-C1-4 alkyl and hydroxy, C1-4 alkoxy-C1-4 alkyl andhalogen, C1-4 alkylthio-C1-4 alkyl and C1-4 alkyl, C1-4 alkylthio-C1-4alkyl and C1-4 alkoxy, C1-4 alkylthio-C1-4 alkyl and hydroxy, or C1-4alkylthio-C1-4 alkyl and halogen, iii) haloalkyl or hydroxy-C1-4 alkyl,or iv) C1-4 alkyl and hydroxy; and

represents a single bond or a double bond, and wherein, when R^(2b) is—O—COR^(8b), the 8-9 position represents a double bond, a salt thereofor a cyclodextrin clathrate thereof.
 36. The method according to claim35, wherein the EP4 agonist is (1) (11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid, or (2)(11α,13E,15α)-9-oxo-11,15-dihydroxy-16-(3-methoxymethylphenyl)-17,18,19,20-tetranor-5-thiaprost-13-enoicacid methyl ester.